US20230083005A1 - Methods of killing or inhibiting the growth of cancer cells - Google Patents

Methods of killing or inhibiting the growth of cancer cells Download PDF

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US20230083005A1
US20230083005A1 US17/798,421 US202117798421A US2023083005A1 US 20230083005 A1 US20230083005 A1 US 20230083005A1 US 202117798421 A US202117798421 A US 202117798421A US 2023083005 A1 US2023083005 A1 US 2023083005A1
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cancer
ptx3
cells
tumor
cell
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Scheffer Tseng
Hua He
Sean Tighe
Kaustuv BASU
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BioTissue Holdings Inc
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BioTissue Holdings Inc
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Publication of US20230083005A1 publication Critical patent/US20230083005A1/en
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Assigned to BIOTISSUE OCULAR INC. (F/K/A BIO-TISSUE, INC.), BIOTISSUE SURGICAL INC. (F/K/A AMNIOX MEDICAL, INC.), BIOTISSUE HOLDINGS INC. (F/K/A TISSUE TECH, INC.) reassignment BIOTISSUE OCULAR INC. (F/K/A BIO-TISSUE, INC.) RELEASE BY SECURED PARTY (SEE DOCUMENT FOR DETAILS). Assignors: MIDCAP FUNDING IV TRUST
Assigned to BIOTISSUE HOLDINGS INC. (F/K/A TISSUE TECH, INC.), BIOTISSUE OCULAR INC. (F/K/A BIO-TISSUE, INC.), BIOTISSUE SURGICAL INC. (F/K/A AMNIOX MEDICAL, INC.) reassignment BIOTISSUE HOLDINGS INC. (F/K/A TISSUE TECH, INC.) RELEASE BY SECURED PARTY (SEE DOCUMENT FOR DETAILS). Assignors: MIDCAP FINANCIAL TRUST
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    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
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    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/61—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule the organic macromolecular compound being a polysaccharide or a derivative thereof
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
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    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
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    • C07K14/8107—Endopeptidase (E.C. 3.4.21-99) inhibitors
    • C07K14/811—Serine protease (E.C. 3.4.21) inhibitors
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
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    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
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    • C07K14/52—Cytokines; Lymphokines; Interferons
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2502/00—Coculture with; Conditioned medium produced by
    • C12N2502/02—Coculture with; Conditioned medium produced by embryonic cells
    • C12N2502/025—Coculture with; Conditioned medium produced by embryonic cells extra-embryonic cells, e.g. amniotic epithelium, placental cells, Wharton's jelly

Definitions

  • the cancer cells are from or within a solid tumor.
  • the cancer cells are from a cancer selected from the group consisting of liver cancer, pancreatic cancer, bladder cancer, prostate cancer, lung cancer, non-small cell lung carcinoma, ovarian cancer, breast cancer, melanoma, gastric cancer, colon cancer, colorectal cancer, central nervous system (CNS) cancer, bone cancer, a lymphoma, skin cancer, head and neck cancer, kidney cancer, testicular cancer, uterine cancer, cervical cancer, esophageal cancer, thyroid cancer, salivary gland cancer, adrenal cancer, and gastrointestinal cancer.
  • CNS central nervous system
  • the cancer is inoperable.
  • the CNS cancer is a glioma or a metastatic cancer.
  • the glioma is glioblastoma multiforme or anaplastic astrocytoma.
  • the cancer is lung cancer, breast cancer, colon cancer or skin cancer.
  • the colon cancer is adenocarcinoma.
  • the skin cancer is melanoma.
  • the cancer is prostate cancer.
  • the contacting comprises injecting the HC-HA/PTX3 complex into the tumor, the surrounding tissue, or a combination thereof.
  • the contacting is prior to, during, or after surgical excision, cryoablation, or radiofrequency ablation of the cancer cells. In some embodiments, the contacting comprises applying the HC-HA/PTX3 to surgical margins from the surgical excision of the cancer cells, or into any remaining portion of tumor.
  • the isolated HC-HA/PTX3 complex is native HC-HA/PTX3 complex, reconstituted HC-HA/PTX3 complex, or a combination thereof. In some embodiments, the native HC-HA/PTX3 complex is isolated from a fetal support tissue.
  • the reconstituted HC-HA/PTX3 complex comprises heavy chain 1 (HC1) and heavy chain 2 (HC2) of inter- ⁇ -inhibitor (I ⁇ I), hyaluronic acid (HA), and PTX3.
  • the reconstituted HC-HA/PTX3 complex comprises HC1, HC2, HA, PTX3 and TSG-6.
  • the HC-HA/PTX3 is comprised in a composition comprising a pharmaceutically acceptable pharmaceutically acceptable diluent, excipient, vehicle, or carrier. In some embodiments, the method further comprising administering a further therapeutic agent.
  • the therapeutic agent is selected from the group consisting of a chemotherapeutic, an analgesic, an anti-inflammatory, a steroid, an immunotherapy, cellular therapy, a radiotherapy, a targeted drug therapeutic, and an antibiotic.
  • administering the therapeutic agent occurs before contacting the cancer cells with the HC-HA/PTX3 complex.
  • administering the therapeutic agent occurs after contacting the cancer cells with the HC-HA/PTX3 complex.
  • administering the therapeutic agent occurs concurrently with contacting the cancer cells with the HC-HA/PTX3 complex.
  • angiogenesis is reduced or inhibited.
  • killing cancer cells is via apoptosis or necrosis.
  • the cancer cells are from or within a solid tumor.
  • the cancer cells are from a cancer selected from the group consisting of liver cancer, pancreatic cancer, bladder cancer, prostate cancer, lung cancer, non-small cell lung carcinoma, ovarian cancer, breast cancer, melanoma, gastric cancer, colon cancer, colorectal cancer, central nervous system (CNS) cancer, bone cancer, a lymphoma, skin cancer, head and neck cancer, kidney cancer, testicular cancer, uterine cancer, cervical cancer, esophageal cancer, thyroid cancer, salivary gland cancer, adrenal cancer, and gastrointestinal cancer.
  • CNS central nervous system
  • the cancer is inoperable.
  • the CNS cancer is a glioma or a metastatic cancer.
  • the glioma is glioblastoma multiforme or anaplastic astrocytoma.
  • the cancer is lung cancer, breast cancer, colon cancer or skin cancer.
  • the colon cancer is adenocarcinoma.
  • the skin cancer is melanoma.
  • the cancer is prostate cancer.
  • the contacting comprises injecting the HC-HA/PTX3 complex into the tumor, the surrounding tissue, or a combination thereof.
  • the contacting is prior to, during, or after surgical excision, cryoablation, or radiofrequency ablation of the cancer cells. In some embodiments, the contacting comprises applying the HC-HA/PTX3 to surgical margins from the surgical excision of the cancer cells.
  • the isolated HC-HA/PTX3 complex is native HC-HA/PTX3 complex, reconstituted HC-HA/PTX3 complex, or a combination thereof. In some embodiments, the native HC-HA/PTX3 complex is isolated from a fetal support tissue.
  • the reconstituted HC-HA/PTX3 complex comprises heavy chain 1 (HC1) and heavy chain 2 (HC2) of inter- ⁇ -inhibitor (I ⁇ I), hyaluronic acid (HA), and PTX3.
  • the reconstituted HC-HA/PTX3 complex comprises HC1, HC2, HA, PTX3 and TSG-6.
  • the HC-HA/PTX3 is comprised in a composition comprising administering a therapeutic agent.
  • the therapeutic agent is selected from the group consisting of a chemotherapeutic, an analgesic, an anti-inflammatory, a steroid, an immunotherapy, cellular therapy, a radiotherapy, a targeted drug therapeutic, and an antibiotic.
  • administering the therapeutic agent occurs before contacting the cancer cells with the HC-HA/PTX3 complex.
  • administering the therapeutic agent occurs after contacting the cancer cells with the HC-HA/PTX3 complex.
  • administering the therapeutic agent occurs concurrently with contacting the cancer cells with the HC-HA/PTX3 complex.
  • angiogenesis is reduced or inhibited.
  • proliferation is inhibited in cells expressing CD44 or RHAMM.
  • the cancer cells are from or within a solid tumor.
  • the cancer cells are from a cancer selected from the group consisting of liver cancer, pancreatic cancer, bladder cancer, prostate cancer, lung cancer, non-small cell lung carcinoma, ovarian cancer, breast cancer, melanoma, gastric cancer, colon cancer, colorectal cancer, central nervous system (CNS) cancer, bone cancer, a lymphoma, skin cancer, head and neck cancer, kidney cancer, testicular cancer, uterine cancer, cervical cancer, esophageal cancer, thyroid cancer, salivary gland cancer, adrenal cancer, and gastrointestinal cancer.
  • CNS central nervous system
  • the cancer is inoperable.
  • the CNS cancer is a glioma or a metastatic cancer.
  • the glioma is glioblastoma multiforme or anaplastic astrocytoma.
  • the cancer is lung cancer, breast cancer, colon cancer or skin cancer.
  • the colon cancer is adenocarcinoma.
  • the skin cancer is melanoma.
  • the cancer is prostate cancer.
  • the contacting comprises injecting the HC-HA/PTX3 complex into the tumor, the surrounding tissue, or a combination thereof.
  • the contacting is prior to, during, or after surgical excision, cryoablation, or radiofrequency ablation of the cancer cells, or into any remaining portion of tumor.
  • the contacting comprises applying the HC-HA/PTX3 to surgical margins from the surgical excision of the cancer cells.
  • the isolated HC-HA/PTX3 complex is native HC-HA/PTX3 complex, reconstituted HC-HA/PTX3 complex, or a combination thereof.
  • the native HC-HA/PTX3 complex is isolated from a fetal support tissue.
  • the reconstituted HC-HA/PTX3 complex comprises heavy chain 1 (HC1) and heavy chain 2 (HC2) of inter- ⁇ -inhibitor (I ⁇ I), hyaluronic acid (HA), and PTX3.
  • the reconstituted HC-HA/PTX3 complex comprises HC1, HC2, HA, PTX3 and TSG-6.
  • the HC-HA/PTX3 is comprised in a composition comprising a pharmaceutically acceptable pharmaceutically acceptable diluent, excipient, vehicle, or carrier.
  • the HC-HA/PTX3 is comprised in a composition comprising administering a therapeutic agent.
  • the therapeutic agent is selected from the group consisting of a chemotherapeutic, an analgesic, an anti-inflammatory, a steroid, an immunotherapy, cellular therapy, a radiotherapy, a targeted drug therapeutic, and an antibiotic.
  • administering the therapeutic agent occurs before contacting the cancer cells with the HC-HA/PTX3 complex.
  • administering the therapeutic agent occurs after contacting the cancer cells with the HC-HA/PTX3 complex.
  • administering the therapeutic agent occurs concurrently with contacting the cancer cells with the HC-HA/PTX3 complex.
  • angiogenesis is reduced or inhibited.
  • metabolic activity is reduced in cells expressing CD44 or RHAMM.
  • Described herein are methods of killing cancer cells, comprising contacting surgical margins or any portion of a tumor prior to, during, or after surgical excision, cryoablation, or radiofrequency ablation of a tumor with isolated HC-HA/PTX3 complex, thereby killing cancer cells in the surgical margins.
  • the tumor is a cancer selected from the group consisting of liver cancer, pancreatic cancer, bladder cancer, prostate cancer, lung cancer, non-small cell lung carcinoma, ovarian cancer, breast cancer, melanoma, gastric cancer, colon cancer, colorectal cancer, central nervous system (CNS) cancer, bone cancer, a lymphoma, skin cancer, head and neck cancer, kidney cancer, testicular cancer, uterine cancer, cervical cancer, esophageal cancer, thyroid cancer, salivary gland cancer, adrenal cancer, and gastrointestinal cancer.
  • the cancer is inoperable.
  • the CNS cancer is a glioma or a metastatic cancer.
  • the glioma is glioblastoma multiforme.
  • the cancer is lung cancer, breast cancer, colon cancer or skin cancer.
  • the colon cancer is adenocarcinoma.
  • the skin cancer is melanoma.
  • the tumor is a prostate cancer.
  • the HC-HA/PTX3 complex is native HC-HA/PTX3 complex, reconstituted HC-HA/PTX3 complex, or a combination thereof.
  • the native HC-HA/PTX3 complex is isolated from a fetal support tissue.
  • the reconstituted HC-HA/PTX3 complex comprises heavy chain 1 (HC1) and heavy chain 2 (HC2) of inter- ⁇ -inhibitor (I ⁇ I), hyaluronic acid (HA), and PTX3.
  • the reconstituted HC-HA/PTX3 complex comprises HC1, HC2, HA, PTX3 and TSG-6.
  • the HC-HA/PTX3 is comprised in a composition comprising a pharmaceutically acceptable pharmaceutically acceptable diluent, excipient, vehicle, or carrier.
  • the reconstituted HC-HA/PTX3 complex comprises administering a therapeutic agent.
  • the therapeutic agent is selected from the group consisting of a chemotherapeutic, an analgesic, an anti-inflammatory, a steroid, an immunotherapy, cellular therapy, a radiotherapy, a targeted drug therapeutic, and an antibiotic.
  • administering the therapeutic agent occurs before contacting the cancer cells with the HC-HA/PTX3 complex.
  • administering the therapeutic agent occurs after contacting the cancer cells with the HC-HA/PTX3 complex.
  • administering the therapeutic agent occurs concurrently with contacting the cancer cells with the HC-HA/PTX3 complex.
  • the killing cancer cells is by apoptosis or necrosis.
  • HC-HA/PTX3 isolated heavy chain-hyaluronan/pentraxin 3
  • the surgical procedure comprises surgical excision, cryoablation, or radiofrequency ablation of the tumor.
  • the surgical procedure comprises chemotherapy, immunotherapy, or targeted therapy.
  • the area surrounding the tumor comprises a surgical margin.
  • the area surrounding the tumor is a peritumor region.
  • the tumor is a solid tumor.
  • the tumor is a cancer selected from the group consisting of liver cancer, pancreatic cancer, bladder cancer, prostate cancer, lung cancer, non-small cell lung carcinoma, ovarian cancer, breast cancer, melanoma, gastric cancer, colon cancer, colorectal cancer, central nervous system (CNS) cancer, bone cancer, a lymphoma, skin cancer, head and neck cancer, kidney cancer, testicular cancer, uterine cancer, cervical cancer, esophageal cancer, thyroid cancer, salivary gland cancer, adrenal cancer, and gastrointestinal cancer.
  • the cancer is an inoperable cancer.
  • the cancer is pancreatic cancer.
  • the cancer is prostate cancer.
  • the cancer is glioblastoma multiforme. In some embodiments, the cancer is skin cancer. In some embodiments, the cancer is colon cancer. In some embodiments, the cancer is lung cancer. In some embodiments, the cancer is breast cancer. In some embodiments, the area surrounding the tumor is contacted with about 10 microgram to 100 milligrams.
  • the HC-HA/PTX3 complex is native HC-HA/PTX3 complex, reconstituted HC-HA/PTX3 complex, or a combination thereof. In some embodiments, the native HC-HA/PTX3 complex is isolated from a fetal support tissue.
  • the reconstituted HC-HA/PTX3 complex comprises heavy chain 1 (HC1) and heavy chain 2 (HC2) of inter- ⁇ -inhibitor (I ⁇ I), hyaluronic acid (HA), and PTX3.
  • the reconstituted HC-HA/PTX3 complex comprises HC1, HC2, HA, PTX3 and tumor necrosis factor ⁇ -stimulated gene 6 (TSG-6).
  • the hyaluronan (HA) is high molecular weight hyaluronan (HMW HA).
  • the hyaluronan (HA) is low molecular weight hyaluronan (LMW HA).
  • the HC-HA/PTX3 complex is cryopreserved. In some embodiments, the HC-HA/PTX3 complex comprises viable cells. In some embodiments, the method further comprises administering a therapeutic agent. In some embodiments, the therapeutic agent is selected from the group consisting of a chemotherapeutic, an analgesic, an anti-inflammatory, a steroid, an immunotherapy, cellular therapy, a radiotherapy, a targeted drug therapeutic, and an antibiotic. In some embodiments, administering the therapeutic agent occurs before contacting the area surrounding the tumor with the HC-HA/PTX3 complex. In some embodiments, administering the therapeutic agent occurs after contacting the area surrounding the tumor with the HC-HA/PTX3 complex.
  • administering the therapeutic agent occurs concurrently with contacting the area surrounding the tumor with the HC-HA/PTX3 complex.
  • the method inhibits tumor cell regrowth by killing cancer cells. In some embodiments, the killing of the cancer cells is by apoptosis or necrosis. In some embodiments, the method inhibits tumor cell regrowth by inhibiting proliferation of cancer cells. In some embodiments, the method inhibits tumor cell regrowth by inhibiting metabolic activity of cancer cells.
  • HC-HA/PTX3 isolated heavy chain-hyaluronan/pentraxin 3
  • the surgical procedure comprises surgical excision, cryoablation, or radiofrequency ablation of the tumor.
  • the surgical procedure comprises chemotherapy, immunotherapy, or targeted therapy.
  • the area surrounding the tumor comprises a surgical margin.
  • the area surrounding the tumor is a peritumor region.
  • the tumor is a solid tumor.
  • the tumor is a cancer selected from the group consisting of liver cancer, pancreatic cancer, bladder cancer, prostate cancer, lung cancer, non-small cell lung carcinoma, ovarian cancer, breast cancer, melanoma, gastric cancer, colon cancer, colorectal cancer, central nervous system (CNS) cancer, bone cancer, a lymphoma, skin cancer, head and neck cancer, kidney cancer, testicular cancer, uterine cancer, cervical cancer, esophageal cancer, thyroid cancer, salivary gland cancer, adrenal cancer, and gastrointestinal cancer.
  • the cancer is an inoperable cancer.
  • the cancer is pancreatic cancer.
  • the cancer is prostate cancer.
  • the cancer is glioblastoma multiforme. In some embodiments, the cancer is skin cancer. In some embodiments, the cancer is colon cancer. In some embodiments, the cancer is lung cancer. In some embodiments, the cancer is breast cancer. In some embodiments, the area surrounding the tumor is contacted with about 10 microgram to 100 milligrams.
  • the HC-HA/PTX3 complex is native HC-HA/PTX3 complex, reconstituted HC-HA/PTX3 complex, or a combination thereof. In some embodiments, the native HC-HA/PTX3 complex is isolated from a fetal support tissue.
  • the reconstituted HC-HA/PTX3 complex comprises heavy chain 1 (HC1) and heavy chain 2 (HC2) of inter- ⁇ -inhibitor (I ⁇ I), hyaluronic acid (HA), and PTX3.
  • the reconstituted HC-HA/PTX3 complex comprises HC1, HC2, HA, PTX3 and tumor necrosis factor ⁇ -stimulated gene 6 (TSG-6).
  • the HC-HA/PTX3 complex is cryopreserved.
  • the HC-HA/PTX3 complex comprises viable cells.
  • the hyaluronan (HA) is high molecular weight hyaluronan (HMW HA).
  • the hyaluronan (HA) is low molecular weight hyaluronan (LMW HA).
  • the contacting comprises injecting the HC-HA/PTX3 directly into a tumor.
  • the method further comprises administering a therapeutic agent.
  • the therapeutic agent is selected from the group consisting of a chemotherapeutic, an analgesic, an anti-inflammatory, a steroid, an immunotherapy, cellular therapy, a radiotherapy, a targeted drug therapeutic, and an antibiotic.
  • administering the therapeutic agent occurs before contacting the cancer cells with the HC-HA/PTX3 complex.
  • administering the therapeutic agent occurs after contacting the cancer cells with the HC-HA/PTX3 complex. In some embodiments, administering the therapeutic agent occurs concurrently with contacting the cancer cells with the HC-HA/PTX3 complex. In some embodiments, the killing of the cancer cells is by apoptosis or necrosis.
  • FIG. 1 A shows aggregation of LNCaP cells after culturing in RPMI medium.
  • FIG. 1 B shows even distribution of PC-3 cells after culturing in RPMI medium.
  • FIG. 2 A - FIG. 2 D show morphology and cell metabolic activity of LNCaP with treatment of a series doses of the refined BTGel or HC-HA/PTX3.
  • FIG. 3 A - FIG. 3 D show morphology and metabolic activity of PC-3 with treatment of a series doses of the refined BTGel or HC-HA/PTX3.
  • FIG. 4 A shows WST-1 assay data in LNCaP cells after treatment with UC extract, HC-HA/PTX3, and HA using UC extract in water.
  • FIG. 4 B shows WST-1 assay data in PC-3 cells after treatment with UC extract, HC-HA/PTX3, and HA using UC extract in water.
  • FIG. 5 A - FIG. 5 C show morphology of LNCaP cells following treatment with HA ( FIG. 5 A ), HC-HA/PTX3 ( FIG. 5 B ), and umbilical cord extract (UCE) ( FIG. 5 C ).
  • FIG. 6 A - FIG. 6 C show morphology of PC-3 cells following treatment with HA ( FIG. 6 A ), HC-HA/PTX3 ( FIG. 6 B ), and umbilical cord extract (UCE) ( FIG. 6 C ).
  • FIG. 7 shows LNCaP cells grown on laminin and collagen type IV exhibit more cell aggregation than cells grown on other surfaces.
  • FIG. 8 A shows bright-field image of PrEC prostate cell line morphology taken in 10 ⁇ and 20 ⁇ magnifications.
  • FIG. 8 B shows bright-field image of PNT2 prostate cell line morphology taken in 10 ⁇ and 20 ⁇ magnifications.
  • FIG. 9 shows representative bright-field microscopic images (scale bar 50 ⁇ m) of human primary normal prostate cells after 48 h incubation with different concentrations of HC-HA/PTX3 and HMW-HA.
  • FIG. 10 A and 10 B shows metabolic activity (%) evaluated in the normal human primary prostate epithelial cells PrEC ( FIG. 10 A ) and normal human prostate cell line PNT2 ( FIG. 10 B ) by WST-1 assay after 48 h incubation with different concentrations (0.78, 3.125, 6.25, 12.5, 25, 50, 100 ⁇ g/ml) of HC-HA/PTX3 and HA. P-value calculated by 2-tailed t-test with respect to the untreated samples.
  • FIG. 11 A and 11 B show comparative analysis of the metabolic activity (%) ( FIG. 11 A ) and log scale metabolic activity ( FIG. 11 B ) evaluated in the normal primary prostate epithelial cells (PrEC) & cell lines (PNT2) and prostate cancer cell lines: PC3 & LNCaP after 48 h incubation with different concentrations (0.78, 3.125, 6.25, 12.5, 25, 50, 100 ⁇ g/ml) of HC-HA/PTX3.
  • FIG. 12 A and 12 B show comparative analysis of the metabolic activity (%) ( FIG. 12 A ) and log scale metabolic activity ( FIG. 12 B ) evaluated in the normal primary prostate epithelial cells (PrEC) & cell lines (PNT2) and prostate cancer cell lines: PC3 & LNCaP after 48 h incubation with different concentrations of HA.
  • FIG. 13 shows representative bright-field microscopic images (scale bar 50 ⁇ m) of A375 (melanoma) cells after 48 h incubation with different concentrations of HC-HA/PTX3 and HMW-HA.
  • FIG. 14 shows representative bright-field microscopic images (scale bar 50 ⁇ m) of HT-29 (colon cancer) cells after 48 h incubation with different concentrations of HC-HA/PTX3 and HMW-HA.
  • FIG. 15 shows representative bright-field microscopic images (scale bar 50 ⁇ m) of A549 (lung cancer) cells after 48 h incubation with different concentrations of HC-HA/PTX3 and HMW-HA.
  • FIG. 16 shows representative bright-field microscopic images (scale bar 50 ⁇ m) of MCF-7 (breast cancer) cells after 48 h incubation with different concentrations of HC-HA/PTX3 and HMW-HA.
  • FIG. 17 A- 17 D show metabolic activity (%) evaluated in 4 human cancer cell lines: A375 ( FIG. 17 A ), HT-29 ( FIG. 17 B ), MCF-7 ( FIG. 17 C ), and A-549 ( FIG. 17 D ) by WST-1 assay after 48 h incubation with different concentrations of HC-HA/PTX3 and HA.
  • FIG. 18 A shows representative bright-field microscopic images (scale bar 50 ⁇ m) of LNC (limbal niche cells) after treatment with different concentrations of HC-HA/PTX3 for different time points: 15-30 min, 1 h, 5 h, 24 h and 48 h respectively.
  • FIG. 18 B shows representative bright-field microscopic images (scale bar 50 ⁇ m) of LNC (limbal niche cells) after treatment with different concentrations of HMW-HA for different time points: 15-30 min, 1 h, 5 h, 24 h and 48 h respectively.
  • FIG. 18 C shows representative bright-field microscopic image (scale bar 50 ⁇ m) of LNC (limbal niche cells) after 48 h incubation with 100 ⁇ g/ml of HC-HA/PTX3 and HMW-HA.
  • FIG. 19 shows metabolic activity (%) evaluated in limbal niche cells by WST-1 assay after 48 h incubation with different concentrations of HC-HA/PTX3 and HA.
  • FIG. 20 A and 20 B show representative bright-field microscopic images (scale bar 50 ⁇ m) of HTM (human trabecular meshwork) cells after treatment with different concentrations of HC-HA/PTX3 ( FIG. 20 A ) and HMW-HA ( FIG. 20 B ) for different time points.
  • FIG. 21 shows metabolic activity (%) evaluated in human trabecular meshwork cells by WST-1 assay after 48 h incubation with different concentrations of HC-HA/PTX3 and HA.
  • FIG. 22 A and 22 B shows representative brightfield microscopic images (scale bar 50 ⁇ m) of human corneal fibroblast (HCF) cells after treated with different concentrations of HC-HA/PTX3 ( FIG. 22 A ) and HMW-HA ( FIG. 22 B ) for different time points.
  • FIG. 23 shows metabolic activity (%) evaluated in human corneal fibroblast cells by WST-1 assay after 48 h incubation with different concentrations of HC-HA/PTX3 and HA.
  • FIG. 24 A and 24 B show comparative analysis of the metabolic activity (%) evaluated in three types of human normal primary mesenchymal cells: HCF, HTM & LNC as evaluated by WST-1 assay after 48 h incubation with different concentrations of HC-HA/PTX3 ( FIG. 24 A ) and HA ( FIG. 24 B ).
  • FIG. 25 provides representative bright-field microscopic images (scale bar 50 ⁇ m) showing transient effect of HC-HA/PTX3 (100 ⁇ g/ml) on the morphology of LNC and HCF cells, without a corresponding effect in HTM cells.
  • FIG. 26 A shows bright-field images of A375 cell morphology following treatment with HC-HA/PTX3 and HA, compared to untreated cells.
  • FIG. 26 B shows BrdU cell proliferation assay curve using A375 cells.
  • FIG. 26 C shows semilog scale BrdU cell proliferation assay curve using A375 cells.
  • FIG. 27 A shows bright-field images of PrEC cell morphology in two magnifications (10 ⁇ & 20 ⁇ ) after treatment with HA or HC-HA/PTX3.
  • FIG. 27 B shows BrdU cell proliferation assay curve following HC-HA/PTX3 treatment.
  • FIG. 27 C shows BrdU cell proliferation assay curve following HA treatment.
  • FIG. 28 A shows brightfield images of PNT2 cell morphology following treatment with HC-HA/PTX3, HMW-HA, or untreated.
  • FIG. 28 B shows BrdU cell proliferation assay curve in PNT2 cells following treatment with HC-HA/PTX3 or HA.
  • FIG. 29 A shows bright-field images of PC3 cell morphology following treatment with HC-HA/PTX3, HMW-HA, or untreated.
  • FIG. 29 B shows BrdU cell proliferation assay curve in PC3 cells following treatment with HC-HA/PTX3 or HA.
  • FIG. 30 A shows bright-field images of LNCaP cell morphology following treatment with HC-HA/PTX3, HMW-HA, or untreated.
  • FIG. 30 B shows BrdU cell proliferation assay curve in LNCaP cells following treatment with HC-HA/PTX3 or HA.
  • ranges and amounts can be expressed as “about” a particular value or range. About also includes the exact amount or value. Hence “about 5 ⁇ g” means “about 5 ⁇ g” and also “5 ⁇ g.” In some embodiments, the term “about” includes an amount that would be expected to be within experimental error. In some embodiments, the term “about” refers to the value +/ ⁇ 20%, 10% or 5% of the value.
  • HC-HA/PTX3 complex or isolated HC-HA/PTX3 refers to native HC-HA/PTX3, reconstituted HC-HA/PTX3 or a combination thereof.
  • a reconstituted HC-HA/PTX3 (rcHC-HA/PTX3) complex is an HC-HA/PTX3 complex that is formed by assembly of the component molecules of the complex in vitro.
  • the rcHC-HA/PTX3 complex comprises HA, HC1 and HC2 of I ⁇ I, and PTX3.
  • the rcHC-HA/PTX3 complex comprises HA, HC1 and HC2 of I ⁇ I, PTX3, and TSG-6.
  • the process of assembling the rcHC-HA/PTX3 includes reconstitution with purified native proteins or molecules isolated from biological sources, recombinant proteins generated by recombinant methods, or synthesis of molecules by in vitro synthesis.
  • the purified native proteins used for assembly of the rcHC-HA/PTX3 are proteins in a complex with other proteins (i.e. a multimer, a multichain protein or other complex).
  • PTX3 is purified as a multimer (e.g. a homomultimer) from a cell and employed for assembly of the rcHC-HA/PTX3 complex.
  • a purified native HC-HA/PTX3 (nHC-HA/PTX3) complex refers to an HC-HA/PTX3 complex that is purified from a biological source such as a cell, a tissue or a biological fluid.
  • the HC-HA/PTX3 is isolated from a fetal support tissue, such as placenta, amniotic membrane, chorion, umbilical cord, or umbilical cord amniotic membrane.
  • the HC-HA/PTX3 is isolated from amniotic membrane.
  • the native HC-HA/PTX3 complex comprises HA, HC1 of I ⁇ I, and PTX3.
  • Such complexes are generally assembled in vivo in a subject or ex vivo in cells, tissues, or biological fluids from a subject, including a human or other animal.
  • hyaluronan As used herein, “hyaluronan,” “hyaluronic acid,” or “hyaluronate” (HA) are used interchangeably to refer to a substantially non-sulfated linear glycosaminoglycan (GAG) with repeating disaccharide units of D-glucuronic acid and N-acetylglucosamine (D-glucuronosyl-N-acetylglucosamine).
  • GAG substantially non-sulfated linear glycosaminoglycan
  • D-glucuronosyl-N-acetylglucosamine D-glucuronosyl-N-acetylglucosamine
  • high molecular weight or “HMW,” as in high molecular weight hyaluronan (HMW HA), is meant to refer to HA that has a weight average molecular weight that is greater than about 500 kilodaltons (kDa), such as, for example, between about 500 kDa and about 10,000 kDa, between about 800 kDa and about 8,500 kDa, between about 1100 kDa and about 5,000 kDa, or between about 1400 kDa and about 3,500 kDa.
  • the HMW HA has a weight average molecular weight of 3000 kDa or greater.
  • the HMW HA has a weight average molecular weight of 3000 kDa. In some embodiments, the HMW HA is Healon® with a weight average molecular weight of about 3000 kDa. In some embodiments, HMW HA has a molecular weight of between about 500 kDa and about 10,000 kDa. In some embodiments, HMW HA has a molecular weight of between about 800 kDa and about 8,500 kDa. In some embodiments, HMW HA has a molecular weight of about 3,000 kDa.
  • low molecular weight or “LMW,” as in low molecular weight hyaluronan (LMW HA), is meant to refer to HA that has a weight average molecular weight that is less than 500 kDa, such as for example, less than about 400 kDa, less than about 300 kDa, less than about 200 kDa, about 200-300 kDa, or about 1-300 kDa.
  • LMW HA low molecular weight hyaluronan
  • pentraxin 3, or PTX3, protein or polypeptide refers to any PTX3 protein, including but not limited to, a recombinantly produced protein, a synthetically produced protein, a native PTX3 protein, and a PTX3 protein extracted from cells or tissues.
  • PTX3 include multimeric forms (e.g. homomultimer) of PTX3, including, but not limited to, dimeric, trimeric, tetrameric, pentameric, hexameric, tetrameric, octameric, and other multimeric forms naturally or artificially produced.
  • HA binding protein As used herein, a “hyaluronan binding protein”, “HA binding protein”, or “HABP” refers to any protein that specifically binds to HA.
  • link module means a hyaluronan-binding domains.
  • biological activity refers to the in vivo activities of an nHC-HA/PTX3 or rcHC-HA/PTX3 complex or physiological responses that result upon in vivo administration of an nHC-HA/PTX3 or rcHC-HA/PTX3 complex or a composition or mixture containing an nHC-HA/PTX3 or rcHC-HA/PTX3 complex.
  • Biological activity thus, encompasses therapeutic effects and pharmaceutical activity of nHC-HA/PTX3 or rcHC-HA/PTX3 complexes and compositions and mixtures thereof.
  • the terms “subject”, “individual” and “patient” are used interchangeably. None of the terms are to be interpreted as requiring the supervision of a medical professional (e.g., a doctor, nurse, physician's assistant, orderly, hospice worker).
  • the subject is any animal, including mammals (e.g., a human or non-human animal) and non-mammals. In one embodiment of the methods and compositions provided herein, the mammal is a human.
  • the terms “treat,” “treating” or “treatment,” and other grammatical equivalents include alleviating, abating or ameliorating one or more symptoms of a disease or condition, ameliorating, preventing or reducing the appearance, severity or frequency of one or more additional symptoms of a disease or condition, ameliorating or preventing the underlying metabolic causes of one or more symptoms of a disease or condition, inhibiting the disease or condition, such as, for example, arresting the development of the disease or condition, relieving the disease or condition, causing regression of the disease or condition, relieving a condition caused by the disease or condition, or inhibiting the symptoms of the disease or condition either prophylactically and/or therapeutically.
  • an rcHC-HA/PTX3 complex, native HC-HA/PTX3 complex, or composition disclosed herein is administered to an individual at risk of developing a particular disorder, predisposed to developing a particular disorder, or to an individual reporting one or more of the physiological symptoms of a disorder.
  • placenta refers to the organ that connects a developing fetus to the maternal uterine wall to allow nutrient uptake, waste elimination, and gas exchange via the maternal blood supply.
  • the placenta is composed of three layers. The innermost placental layer surrounding the fetus is called amnion.
  • the allantois is the middle layer of the placenta (derived from the embryonic hindgut); blood vessels originating from the umbilicus traverse this membrane.
  • the outermost layer of the placenta, the chorion comes into contact with the endometrium. The chorion and allantois fuse to form the chorioallantoic membrane.
  • chorion refers to the membrane formed by extraembryonic mesoderm and the two layers of trophoblast.
  • the chorion consists of two layers: an outer formed by the trophoblast, and an inner formed by the somatic mesoderm; the amnion is in contact with the latter.
  • the trophoblast is made up of an internal layer of cubical or prismatic cells, the cytotrophoblast or layer of Langhans, and an external layer of richly nucleated protoplasm devoid of cell boundaries, the syncytiotrophoblast.
  • the avascular amnion is adherent to the inner layer of the chorion.
  • amnion-chorion refers to a product comprising amnion and chorion.
  • the amnion and the chorion are not separated (i.e., the amnion is naturally adherent to the inner layer of the chorion).
  • the amnion is initially separated from the chorion and later combined with the chorion during processing.
  • umbilical cord refers to the organ that connects a developing fetus to the placenta.
  • the umbilical cord is composed of Wharton's jelly, a gelatinous substance made largely from mucopolysaccharides. It contains one vein, which carries oxygenated, nutrient-rich blood to the fetus, and two arteries that carry deoxygenated, nutrient-depleted blood away.
  • placental amniotic membrane refers to amniotic membrane derived from the placenta. In some embodiments, the PAM is substantially isolated.
  • UCAM amniotic membrane derived from the umbilical cord.
  • UCAM is a translucent membrane.
  • the UCAM has multiple layers: an epithelial layer; a basement membrane; a compact layer; a fibroblast layer; and a spongy layer. It lacks blood vessels or a direct blood supply.
  • the UCAM comprises Wharton's Jelly.
  • the UCAM comprises blood vessels and/or arteries.
  • the UCAM comprises Wharton's Jelly and blood vessels and/or arteries.
  • purified mean a material (e.g., nHC-HA/PTX3 complex) substantially or essentially free from components that normally accompany it in its native state.
  • purified or isolated mean a material (e.g., nHC-HA/PTX3 complex) is about 50% or more free from components that normally accompany it in its native state, for example, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% free from components that normally accompany it in its native state.
  • nHC-HA/PTX3 or rcHC-HA/PTX3 complexes described herein including methods to directly kill cancer cells, directly inhibit proliferation of cancer cells, reduce the metabolic activity of cancer cells or a combination thereof.
  • an nHC-HA/PTX3 or rcHC-HA/PTX3 complex is used to directly kill cancer cells.
  • an nHC-HA/PTX3 or rcHC-HA/PTX3 complex is used to directly inhibit proliferation of cancer cells.
  • an nHC-HA/PTX3 or rcHC-HA/PTX3 complex is used to reduce the metabolic activity of cancer cells.
  • cells are from or within a solid tumor.
  • the methods of treating an individual in need thereof comprising administering to the individual nHC-HA/PTX3 or rcHC-HA/PTX3 complexes described herein by any suitable method.
  • the individual in need thereof has cancer.
  • the individual in need thereof has an inoperable cancer.
  • the individual in need thereof has an inoperable cancer selected from the group consisting of pancreatic cancer, prostate cancer, and glioblastoma multiforme.
  • the individual has a solid tumor.
  • the individual has a cancer selected from the group consisting of liver cancer, pancreatic cancer, bladder cancer, prostate cancer, lung cancer, non-small cell lung carcinoma, ovarian cancer, breast cancer, melanoma, gastric cancer, colon cancer, colorectal cancer, central nervous system (CNS) cancer, bone cancer, a lymphoma, skin cancer, head and neck cancer, kidney cancer, testicular cancer, uterine cancer, cervical cancer, esophageal cancer, thyroid cancer, salivary gland cancer, adrenal cancer, and gastrointestinal cancer.
  • a cancer selected from the group consisting of liver cancer, pancreatic cancer, bladder cancer, prostate cancer, lung cancer, non-small cell lung carcinoma, ovarian cancer, breast cancer, melanoma, gastric cancer, colon cancer, colorectal cancer, central nervous system (CNS) cancer, bone cancer, a lymphoma, skin cancer, head and neck cancer, kidney cancer, testicular cancer, uterine cancer, cervical cancer, esophageal cancer, thyroid cancer, salivary gland cancer, adrenal
  • the nHC-HA/PTX3 or rcHC-HA/PTX3 complex is administered to an individual, prior to, during a surgical procedure, or after a surgical procedure.
  • the surgical procedure comprises excision of a tumor.
  • the surgical procedure comprises surgical excision, cryoablation, or radiofrequency ablation of a tumor.
  • the surgical procedure comprises chemotherapy, immunotherapy, or targeted therapy.
  • the nHC-HA/PTX3 or rcHC-HA/PTX3 complex is administered up to 1 day, up to 2 days, up to 3 days, up to 5 days, or more than 5 days following excision of a tumor. In some embodiments, the nHC-HA/PTX3 or rcHC-HA/PTX3 complex is administered one week, two weeks, one month, two months, three months, four months, five months, one year, two years, three years, four years, five years, or more than five years following excision of a tumor.
  • the nHC-HA/PTX3 or rcHC-HA/PTX3 complex is administered singly, or over a time course, such as daily, multiple times weekly, weekly, biweekly, monthly or less frequently following excision of a tumor. In some embodiments, the nHC-HA/PTX3 or rcHC-HA/PTX3 complex is administered singly, or over a time course, such as daily, multiple times weekly, weekly, biweekly, monthly or more frequently following excision of a tumor.
  • the nHC-HA/PTX3 or rcHC-HA/PTX3 complex is administered prior to excision of a tumor. In some embodiments, the nHC-HA/PTX3 or rcHC-HA/PTX3 complex is administered up to 1 day, up to 2 days, up to 3 days, up to 5 days, or more than 5 days prior to excision of a tumor. In some embodiments, the nHC-HA/PTX3 or rcHC-HA/PTX3 complex is administered singly, or over a time course, such as daily, multiple times weekly, weekly, biweekly, monthly or less frequently prior to excision of a tumor.
  • the nHC-HA/PTX3 or rcHC-HA/PTX3 complex is administered singly, or over a time course, such as daily, multiple times weekly, weekly, biweekly, monthly or more frequently prior to excision of a tumor.
  • the nHC-HA/PTX3 or rcHC-HA/PTX3 complex is administered to an individual during or after surgical excision, cryoablation, or radiofrequency ablation of a tumor.
  • the nHC-HA/PTX3 or rcHC-HA/PTX3 complex is administered to a surgical margin (e.g., an area of apparently tissue around a tumor that has been surgically removed) after surgical excision (e.g., whole or partial), cryoablation, or radiofrequency ablation of a tumor.
  • the methods of treating an individual in need thereof comprising administering to the individual nHC-HA/PTX3 or rcHC-HA/PTX3 complexes by any suitable route of administration. Suitable methods for administration will depend on the disease or condition to be treated.
  • the nHC-HA/PTX3 or rcHC-HA/PTX3 complexes are administered locally to the site of treatment.
  • the nHC-HA/PTX3 or rcHC-HA/PTX3 complexes are injected into a tumor, the tissue surrounding the tumor or both.
  • the nHC-HA/PTX3 or rcHC-HA/PTX3 complexes are injected into a tumor.
  • the nHC-HA/PTX3 or rcHC-HA/PTX3 complexes are applied to the area surrounding a tumor after the tumor has been surgically removed or treated with cryoablation or radiofrequency ablation. In some embodiments, the nHC-HA/PTX3 or rcHC-HA/PTX3 complexes are administered systemically. Exemplary methods for administration of the nHC-HA/PTX3 or rcHC-HA/PTX3 complexes provided herein include but are not limited to parenteral, enteral, subcutaneous, percutaneous, transdermal, intradermal, intravenous, topical, inhalation, or implantation.
  • the isolated HC-HA/PTX3 complex is demonstrated herein to directly kill cancer cells.
  • uses of an isolated HC-HA/PTX3 complex including preparations or compositions comprising HC-HA/PTX3, to kill cancer cells.
  • uses of an isolated HC-HA/PTX3 complex to kill cancer cells in a solid tumor.
  • uses of an isolated HC-HA/PTX3 complex to kill cancer cells in the area surrounding a tumor after the tumor has been surgically removed or treated with cryoablation or radiofrequency ablation.
  • provided herein are uses of an isolated HC-HA/PTX3 complex to kill cancer cells locally in a subject in need thereof.
  • the isolated HC-HA/PTX3 complex is demonstrated herein to inhibit proliferation of cancer cells.
  • uses of an isolated HC-HA/PTX3 complex including preparations or compositions comprising HC-HA/PTX3, to inhibit proliferation of cancer cells.
  • uses of an isolated HC-HA/PTX3 complex to inhibit the proliferation of cancer cells in a solid tumor.
  • provided herein are uses of an isolated HC-HA/PTX3 complex to inhibit the proliferation of cancer cells locally in a subject in need thereof. In some embodiments, provided herein are uses of an isolated HC-HA/PTX3 complex to inhibit the proliferation of cancer cells systemically in a subject in need thereof. In some embodiments, proliferation is inhibited or decreased by 5-95%, 10-90%, 20-80%, 30-70%, 40-60%, 50-95%, 65-85%, or 75-95%. In some embodiments, proliferation is inhibited or decreased by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or greater than 95%. In some embodiments, proliferation is inhibited or decreased by at least 5%. In some embodiments, proliferation is inhibited or decreased by at least 10%. In some embodiments, proliferation is inhibited or decreased by at least 50%.
  • the isolated HC-HA/PTX3 complex is demonstrated herein to reduce metabolic activity of cancer cells.
  • uses of an isolated HC-HA/PTX3 complex including preparations or compositions comprising HC-HA/PTX3, to reduce metabolic activity in cancer cells.
  • uses of an isolated HC-HA/PTX3 complex to reduce metabolic activity of cancer cells in a solid tumor.
  • provided herein are uses of an isolated HC-HA/PTX3 complex to reduce metabolic activity of cancer cells locally in a subject in need thereof. In some embodiments, provided herein are uses of an isolated HC-HA/PTX3 complex to reduce metabolic activity of cancer cells systemically in a subject in need thereof. In some embodiments, metabolic activity is reduced by 5-95%, 10-90%, 20-80%, 30-70%, 40-60%, 50-95%, 65-85%, or 75-95%. In some embodiments, metabolic activity is reduced by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or greater than 95%. In some embodiments, metabolic activity is reduced by at least 5%. In some embodiments, metabolic activity is reduced by at least 10%. In some embodiments, metabolic activity is reduced by at least 50%.
  • cancer cell death is increased by about 10% to about 25%, about 10% to about 50%, about 20% to about 90%. In some embodiments, cancer cell death is increased by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or greater than 95%. In some embodiments, cancer cell death is caused by apoptosis. In some embodiments, cancer cell death is caused by necrosis.
  • the cancer cells are from or within a solid tumor.
  • the solid tumor is a liver cancer, pancreatic cancer, bladder cancer, prostate cancer, lung cancer, non-small cell lung carcinoma, ovarian cancer, breast cancer, melanoma, gastric cancer, colon cancer, colorectal cancer, central nervous system (CNS) cancer, bone cancer, a lymphoma, skin cancer, head and neck cancer, kidney cancer, testicular cancer, uterine cancer, cervical cancer, esophageal cancer, thyroid cancer, salivary gland cancer, adrenal cancer, or a gastrointestinal cancer.
  • the cancer cells are from a liquid tumor.
  • the liquid cancer is a lymphoma or leukemia.
  • the CNS cancer is a glioma or a metastatic cancer.
  • the glioma is glioblastoma multiforme or anaplastic astrocytoma.
  • the colon cancer is adenocarcinoma, a carcinoid tumor, a primary colorectal lymphoma, a stromal tumor, or a leiomyosarcoma.
  • the skin cancer is a melanoma, a basal cell carcinoma, or a squamous cell carcinoma.
  • isolated native HC-HA/PTX3 (nHC-HA/PTX3) complexes are used in the methods provided herein.
  • the isolated nHC-HA/PTX3 complex is isolated from an amniotic tissue. In some embodiments, the isolated nHC-HA/PTX3 complex is isolated from an amniotic membrane or an umbilical cord. In some embodiments, the isolated nHC-HA/PTX3 complex is isolated from fresh, frozen or previously frozen placental amniotic membrane (PAM), fresh, frozen or previously frozen umbilical cord amniotic membrane (UCAM), fresh, frozen or previously frozen placenta, fresh, frozen or previously frozen umbilical cord, fresh, frozen or previously frozen chorion, fresh, frozen or previously frozen amnion-chorion, or any combinations thereof.
  • PAM fresh, frozen or previously frozen placental amniotic membrane
  • UCAM fresh, frozen or previously frozen umbilical cord amniotic membrane
  • Such tissues can be obtained from any mammal, such as, for example, but not limited to a human, non-human primate, cow or pig.
  • the nHC-HA/PTX3 is purified by any suitable method.
  • the nHC-HA/PTX3 complex is purified by centrifugation (e.g., ultracentrifugation, gradient centrifugation), chromatography (e.g., ion exchange, affinity, size exclusion, and hydroxyapatite chromatography), tangential flow filtration (TFF), gel filtration, or differential solubility, ethanol precipitation or by any other available technique for the purification of proteins (See, e.g., Scopes, Protein Purification Principles and Practice 2nd Edition, Springer-Verlag, New York, 1987; Higgins, S. J. and Hames, B. D.
  • the nHC-HA/PTX3 is isolated from an extract.
  • the extract is prepared from an amniotic membrane extract.
  • the extract is prepared from an umbilical cord extract.
  • the umbilical cord extract comprises umbilical cord stroma and/or Wharton's jelly.
  • the nHC-HA/PTX3 complex is contained in an extract that is prepared by ultracentrifugation.
  • the nHC-HA/PTX3 complex is contained in an extract that is prepared by ultracentrifugation using a CsCl/4-6 M guanidine HCl gradient.
  • the extract is prepared by at least 2 rounds of ultracentrifugation.
  • the extract is prepared by more than 2 rounds of ultracentrifugation (i.e. nHC-HA/PTX3 2 nd ). In some embodiments, the extract is prepared by at least 4 rounds of ultracentrifugation (i.e. nHC-HA/PTX3 4 th ). In some embodiments, the nHC-HA/PTX3 complex comprises a small leucine-rich proteoglycan. In some embodiments, the nHC-HA/PTX3 complex comprises HC1, HA, PTX3 and/or a small leucine-rich proteoglycan.
  • ultracentrifugation is performed on an extract prepared by extraction in an isotonic solution.
  • the isotonic solution is PBS.
  • the tissue is homogenized in PBS to produce a homogenized sample.
  • the homogenized sample is then separated into a soluble portion and insoluble portion by centrifugation.
  • ultracentrifugation is performed on the soluble portion of the PBS-extracted tissue.
  • the nHC-HA/PTX3 purified by ultracentrifugation of the PBS-extracted tissue called an nHC-HA/PTX3 soluble complex.
  • the nHC-HA soluble complex comprises a small leucine-rich proteoglycan. In some embodiments, the nHC-HA/PTX3 soluble complex comprises HC1, HA, PTX3 and/or a small leucine-rich proteoglycan.
  • ultracentrifugation is performed on an extract prepared by direct guanidine HCl extraction (e.g. 4-6 M GnHCl) of the amniotic membrane and/or umbilical cord tissue.
  • the GnHCl extract tissues is then centrifuged to produce GnHCl soluble and GnHCl insoluble portions.
  • ultracentrifugation is performed on the GnHCl soluble portion.
  • the nHC-HA/PTX3 purified by ultracentrifugation of the guanidine HCl-extracted tissue is called an nHC-HA/PTX3 insoluble complex.
  • the nHC-HA insoluble complex comprises a small leucine-rich proteoglycan. In some embodiments, the nHC-HA/PTX3 insoluble complex comprises HC1, HA, PTX3 and/or a small leucine-rich proteoglycan.
  • ultracentrifugation is performed on an extract prepared by further guanidine HCl extraction of the insoluble portion of the PBS-extracted tissue.
  • the tissue is homogenized in PBS to produce a homogenized sample.
  • the homogenized sample is then separated into a soluble portion and insoluble portion by centrifugation.
  • the insoluble portion is then further extracted in guanidine HCl (e.g. 4-6 M GnHCl) and centrifuged to produce a guanidine HCl soluble and insoluble portions.
  • ultracentrifugation is performed on the guanidine HCl soluble portion.
  • the nHC-HA/PTX3 purified by ultracentrifugation of the guanidine HCl-extracted tissue is called an nHC-HA/PTX3 insoluble complex.
  • the nHC-HA insoluble complex comprises a small leucine-rich proteoglycan.
  • the nHC-HA/PTX3 insoluble complex comprises HC1, HA, PTX3 and/or a small leucine-rich proteoglycan.
  • the method of purifying the isolated nHC-HA/PTX3 extract comprises: (a) dissolving the isolated extract (e.g. prepared by the soluble or insoluble method described herein) in CsCl/4-6 M guanidine HCl at the initial density of 1.35 g/ml, to generate a CsCl mixture, (b) centrifuging the CsCl mixture at 125,000 ⁇ g for 48 h at 15° C.
  • the method of purifying the isolated extract further comprises (d) mixing the dialysate with 3 volumes of 95% (v/v) ethanol containing 1.3% (w/v) potassium acetate at 0° C.
  • the method of purifying the isolated extract further comprises: (g) washing the second purified extract with ethanol (e.g., 70% ethanol), to generate a second purified extract/ethanol mixture; (h) centrifuging the second purified extract/ethanol mixture, to generate a third purified extract; and (i) extracting the third purified extract.
  • ethanol e.g., 70% ethanol
  • the method of purifying the isolated extract further comprises: (j) washing the third purified extract with ethanol (e.g., 70% ethanol), to generate a third purified extract/ethanol mixture; (k) centrifuging the third purified extract/ethanol mixture, to generate a forth purified extract; and (l) extracting the forth purified extract.
  • the purified extract comprises an nHC-HA/PTX3 complex.
  • the nHC-HA/PTX3 complex is purified by immunoaffinity chromatography.
  • anti HC1 antibodies, anti-HC2 antibodies, or both are generated and affixed to a stationary support.
  • the unpurified HC-HA complex i.e., the mobile phase
  • the HC-HA complex binds to the antibodies (e.g., via interaction of (a) an anti-HC1 antibody and HC1, (b) an anti-HC2 antibody and HC2, (c) an anti-PTX antibody and PTX3, (d) an anti-SLRP antibody and the SLRP, or (e) any combination thereof).
  • the support is washed (e.g., with PBS) to remove any unbound or loosely bound molecules.
  • the support is then washed with a solution that enables elution of the nHC-HA/PTX3 complex from the support (e.g., 1% SDS, 6 M guanidine-HCl, or 8 M urea).
  • the nHC-HA/PTX3 complex is purified by affinity chromatography.
  • HABP is generated and affixed to a stationary support.
  • the unpurified nHC-HA/PTX3 complex i.e., the mobile phase
  • the nHC-HA/PTX3 complex binds to the HABP.
  • the support is washed (e.g., with PBS) to remove any unbound or loosely bound molecules.
  • the support is then washed with a solution that enables elution of the HC-HA complex from the support.
  • the nHC-HA/PTX3 complex is purified by a combination of HABP affinity chromatography, and immunoaffinity chromatography using anti HC1 antibodies, anti-HC2 antibodies, anti-PTX3 antibodies, antibodies against a SLRP or a combination of SLRPs, or any combination of antibodies thereof.
  • the nHC-HA/PTX3 complex is purified from the insoluble fraction as described herein using one or more antibodies. In some embodiments, the nHC-HA/PTX3 complex is purified from the insoluble fraction as described herein using anti-SLRP antibodies.
  • the nHC-HA/PTX3 complex is purified from the soluble fraction as described herein. In some embodiments, the nHC-HA/PTX3 complex is purified from the soluble fraction as described herein using anti-PTX3 antibodies.
  • the nHC-HA/PTX3 complex comprises a small leucine rich proteoglycan (SLRP).
  • SLRP small leucine rich proteoglycan
  • the nHC-HA/PTX3 complex comprises a class I, class II or class III SLRP.
  • the small leucine-rich proteoglycan is selected from among class I SLRPs, such as decorin and biglycan.
  • the small leucine-rich proteoglycan is selected from among class II SLRPs, such as fibromodulin, lumican, PRELP (proline arginine rich end leucine-rich protein), keratocan, and osteoadherin.
  • the small leucine-rich proteoglycan is selected from among class III SLRPs, such as epipycan and osteoglycin. In some embodiments, the small leucine-rich proteoglycan is selected from among bikunin, decorin, biglycan, and osteoadherin. In some embodiments, the small leucine-rich protein comprises a glycosaminoglycan. In some embodiments, the small leucine-rich proteoglycan comprises keratan sulfate.
  • rcHC-HA/PTX3 complexes are used in the methods provided herein.
  • Such reconstituted HC-HA/PTX3 complexes can be with or without SLRPs.
  • a method for generating reconstituted HC-HA/PTX3 complexes comprises (a) contacting hyaluronan (HA) with I ⁇ I and TSG-6 to form an HC-HA complex pre-bound to TSG-6 and (b) contacting the HC-HA complex with pentraxin 3 (PTX3) under suitable conditions to form an rcHC-HA/PTX3 complex.
  • PTX3 pentraxin 3
  • rcHC-HA/PTX3 complexes produced by such method.
  • HC1 of I ⁇ I forms a covalent linkage with HA.
  • the steps (a) and (b) of the method are performed sequentially in order.
  • the method comprises contacting an HC-HA complex pre-bound to TSG-6 with PTX3.
  • the hyaluronan (HA) is high molecular weight hyaluronan (HMW HW).
  • the hyaluronan (HA) is low molecular weight hyaluronan (LMW HW).
  • a method for generating reconstituted HC-HA/PTX3 complexes comprises (a) contacting high molecular weight hyaluronan (HMW HA) with I ⁇ I and TSG-6 to form an HC-HA complex pre-bound to TSG-6 and (b) contacting the HC-HA complex with pentraxin 3 (PTX3) under suitable conditions to form an rcHC-HA/PTX3 complex.
  • HMW HA high molecular weight hyaluronan
  • PTX3 pentraxin 3
  • rcHC-HA/PTX3 complexes produced by such method.
  • HC1 of I ⁇ I forms a covalent linkage with HA.
  • the steps (a) and (b) of the method are performed sequentially in order.
  • the method comprises contacting an HC-HA complex pre-bound to TSG-6 with PTX3.
  • the I ⁇ I protein and TSG-6 protein are contacted to the HMW HA at a molar ratio of about 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 15:1, or 20:1 (I ⁇ I:TSG-6).
  • the ratio of I ⁇ I:TSG-6 ranges from about 1:1 to about 20:1, such as about 1:1 to about 10:1, such as about 1:1 to 5 about:1, such as about 1:1 to about 3:1.
  • the ratio of I ⁇ I:TSG-6 is 3:1 or higher.
  • the ratio of I ⁇ I:TSG-6 is 3:1.
  • TSG-6 interacts with I ⁇ I and forms covalent complexes with HC1 and HC2 of I ⁇ I (e.g., HC1 ⁇ TSG-6 and HC2 ⁇ TSG-6).
  • HC1 ⁇ TSG-6 and HC2 ⁇ TSG-6 covalent complexes with HC1 and HC2 of I ⁇ I
  • the HCs are transferred to HA to form rcHC-HA.
  • the step of contacting hyaluronan (HA) with I ⁇ I and TSG-6 occurs for at least 10 minutes, at least 30 minutes, at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 6 hours, at least 12 hours, or at least 24 hours or longer. In some embodiments, the step of contacting HA with I ⁇ I and TSG-6 occurs for at least 2 hours or longer. In some embodiments, the step of contacting HA with I ⁇ I and TSG-6 occurs for at least 2 hours. In some embodiments, the step of contacting HA with I ⁇ I and TSG-6 occurs at 37° C.
  • the step of contacting immobilized HA with I ⁇ I and TSG-6 occurs in 5 mM MgCl 2 in PBS.
  • the hyaluronan (HA) is high molecular weight hyaluronan (HMW HW).
  • the hyaluronan (HA) is low molecular weight hyaluronan (LMW HW).
  • the step of contacting high molecular weight hyaluronan (HMW HA) with I ⁇ I and TSG-6 occurs for at least 10 minutes, at least 30 minutes, at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 6 hours, at least 12 hours, or at least 24 hours or longer. In some embodiments, the step of contacting HMW HA with I ⁇ I and TSG-6 occurs for at least 2 hours or longer. In some embodiments, the step of contacting HMW HA with I ⁇ I and TSG-6 occurs for at least 2 hours. In some embodiments, the step of contacting HMW HA with I ⁇ I and TSG-6 occurs at 37° C. In some embodiments, the step of contacting immobilized HMW HA with I ⁇ I and TSG-6 occurs in 5 mM MgCl 2 in PBS.
  • the step of contacting PTX3 to an HC-HA complex occurs for at least 10 minutes, at least 30 minutes, at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 6 hours, at least 12 hours, or at least 24 hours or longer. In some embodiments, the step of contacting PTX3 to an HC-HA complex occurs for at least 2 hours or longer. In some embodiments, the step of contacting PTX3 to an HC-HA complex occurs for at least 2 hours. In some embodiments, the step of contacting PTX3 to an HC-HA complex occurs at 37° C. In some embodiments, the step of contacting PTX3 to an HC-HA complex occurs in 5 mM MgCl 2 in PBS.
  • the method comprises contacting hyaluronan (HA) with a pentraxin 3 (PTX3) protein, inter- ⁇ -inhibitor (I ⁇ I) protein comprising heavy chain 1 (HC1) and heavy chain 2 (HC2), and Tumor necrosis factor ⁇ -stimulated gene 6 (TSG-6) simultaneously under suitable conditions to form a HC-HA/PTX3 complex.
  • PTX3 pentraxin 3
  • I ⁇ I inter- ⁇ -inhibitor
  • TSG-6 Tumor necrosis factor ⁇ -stimulated gene 6
  • the step of contacting the HA, PTX3, I ⁇ I, and TSG-6 occurs at 37° C. In some embodiments the step of contacting the HA, PTX3, I ⁇ I, and TSG-6 occurs in 5 mM MgCl 2 in PBS.
  • the hyaluronan (HA) is high molecular weight hyaluronan (HMW HW). In some embodiments, the hyaluronan (HA) is low molecular weight hyaluronan (LMW HW).
  • the method comprises contacting high molecular weight hyaluronan (HMW HA) with a pentraxin 3 (PTX3) protein, inter- ⁇ -inhibitor (I ⁇ I) protein comprising heavy chain 1 (HC1) and heavy chain 2 (HC2), and Tumor necrosis factor ⁇ -stimulated gene 6 (TSG-6) simultaneously under suitable conditions to form a HC-HA/PTX3 complex.
  • HMW HA high molecular weight hyaluronan
  • PTX3 pentraxin 3
  • I ⁇ I inter- ⁇ -inhibitor
  • TSG-6 Tumor necrosis factor ⁇ -stimulated gene 6
  • the step of contacting the HMW HA, PTX3, I ⁇ I, and TSG-6 occurs at 37° C. In some embodiments the step of contacting the HMW HA, PTX3, I ⁇ I, and TSG-6 occurs in 5 mM MgCl 2 in PBS.
  • the method comprises contacting hyaluronan (HA) with a pentraxin 3 (PTX3) protein, inter- ⁇ -inhibitor (I ⁇ I) protein comprising heavy chain 1 (HC1) and heavy chain 2 (HC2), and Tumor necrosis factor ⁇ -stimulated gene 6 (TSG-6) sequentially, in any order, under suitable conditions to form a HC-HA/PTX3 complex.
  • PTX3 pentraxin 3
  • I ⁇ I inter- ⁇ -inhibitor
  • TSG-6 Tumor necrosis factor ⁇ -stimulated gene 6
  • the step of contacting the HA, PTX3, I ⁇ I, and TSG-6 occurs at 37° C. In some embodiments the step of contacting the HA, PTX3, I ⁇ I, and TSG-6 occurs in 5 mM MgCl 2 in PBS.
  • the hyaluronan (HA) is high molecular weight hyaluronan (HMW HW). In some embodiments, the hyaluronan (HA) is low molecular weight hyaluronan (LMW HW).
  • the method comprises contacting high molecular weight hyaluronan (HMW HA) with a pentraxin 3 (PTX3) protein, inter- ⁇ -inhibitor (I ⁇ I) protein comprising heavy chain 1 (HC1) and heavy chain 2 (HC2), and Tumor necrosis factor ⁇ -stimulated gene 6 (TSG-6) sequentially, in any order, under suitable conditions to form a HC-HA/PTX3 complex.
  • HMW HA high molecular weight hyaluronan
  • PTX3 pentraxin 3
  • I ⁇ I inter- ⁇ -inhibitor
  • TSG-6 Tumor necrosis factor ⁇ -stimulated gene 6
  • the contacting the HMW HA with PTX3, I ⁇ I and TSG-6 occurs for at least 10 minutes, at least 30 minutes, at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 6 hours, at least 12 hours, or at least 24 hours or longer.
  • the step of contacting the HMW HA, PTX3, I ⁇ I, and TSG-6 occurs at 37° C. In some embodiments the step of contacting the HMW HA, PTX3, I ⁇ I, and TSG-6 occurs in 5 mM MgCl 2 in PBS.
  • the methods for production of an rcHC-HA/PTX3 complex further comprises addition of one or more small leucine rich proteoglycans (SLRPs).
  • a method for generating reconstituted HC-HA/PTX3 complexes comprises (a) contacting hyaluronan (HA) with I ⁇ I and TSG-6 to HA to form an HC-HA complex pre-bound to TSG-6, (b) contacting the HC-HA complex with pentraxin 3 (PTX3) and (c) contacting the HC-HA complex with one or more SLRPS under suitable conditions to form an rcHC-HA/PTX3 complex.
  • HA hyaluronan
  • PTX3 pentraxin 3
  • a method for generating reconstituted HC-HA/PTX3 complexes comprises (a) contacting high molecular weight hyaluronan (HMW HA) with I ⁇ I and TSG-6 to HA to form an HC-HA complex pre-bound to TSG-6, (b) contacting the HC-HA complex with pentraxin 3 (PTX3) and (c) contacting the HC-HA complex with one or more SLRPS under suitable conditions to form an rcHC-HA/PTX3 complex.
  • HMW HA high molecular weight hyaluronan
  • PTX3 pentraxin 3
  • HC-HA complex with one or more SLRPS under suitable conditions to form an rcHC-HA/PTX3 complex.
  • HC1 of I ⁇ I forms a covalent linkage with HA.
  • the method comprises contacting an HC-HA complex pre-bound to TSG-6 with PTX3.
  • the steps (a), (b), and (c) of the method are performed sequentially in order.
  • the steps (a), (b), and (c) of the method are performed simultaneously.
  • the step (a) of the method is performed and then steps (b) and (c) of the method are performed sequentially in order.
  • the step (a) of the method is performed and then steps (b) and (c) of the method are performed simultaneously.
  • the SLRP is selected from among a class I, class II or class II SLRP. In some embodiments, the SLRP is selected from among class I SLRPs, such as decorin and biglycan. In some embodiments, the small leucine-rich proteoglycan is selected from among class II SLRPs, such as fibromodulin, lumican, PRELP (proline arginine rich end leucine-rich protein), keratocan, and osteoadherin. In some embodiments, the small leucine-rich proteoglycan is selected from among class III SLRPs, such as epipycan and osteoglycin.
  • the small leucine-rich proteoglycan is selected from among bikunin, decorin, biglycan, and osteoadherin.
  • the small leucine-rich protein comprises a glycosaminoglycan.
  • the small leucine-rich proteoglycan comprises keratan sulfate.
  • PTX3 for use in the methods is isolated from a cell or a plurality of cells (e.g., a tissue extract).
  • Exemplary cells suitable for the expression of PTX3 include, but are not limited to, animal cells including, but not limited to, mammalian cells, primate cells, human cells, rodent cells, insect cells, bacteria, and yeast, and plant cells, including, but not limited to, algae, angiosperms, gymnosperms, pteridophytes and bryophytes.
  • PTX3 for use in the methods is isolated from a human cell.
  • PTX3 for use in the methods is isolated from a cell that is stimulated with one or more proinflammatory cytokines to upregulate PTX3 expression.
  • the proinflammatory cytokine is IL-1 or TNF- ⁇ .
  • PTX3 for use in the methods is isolated from an amniotic membrane cell. In some embodiments, PTX3 for use in the methods is isolated from an amniotic membrane cell from an umbilical cord. In some embodiments, the amniotic membrane cell is stimulated with or more proinflammatory cytokines to upregulate PTX3 expression. In some embodiments, the proinflammatory cytokine is IL-1 or TNF- ⁇ .
  • PTX3 for use in the methods is isolated from an umbilical cord cell.
  • the umbilical cord cell is stimulated with or more proinflammatory cytokines to upregulate PTX3 expression.
  • the proinflammatory cytokine is IL-1 or TNF- ⁇ .
  • PTX3 for use in the methods is isolated from an amniotic epithelial cell. In some embodiments, PTX3 for use in the methods is isolated from an umbilical cord epithelial cell. In some embodiments, the amniotic epithelial cell or umbilical cord epithelial cell is stimulated with or more proinflammatory cytokines to upregulate PTX3 expression. In some embodiments, the proinflammatory cytokine is IL-1 or TNF- ⁇ .
  • PTX3 for use in the methods is isolated from an amniotic stromal cell. In some embodiments, PTX3 for use in the methods is isolated from an umbilical cord stromal cell. In some embodiments, the amniotic stromal cell or umbilical cord stromal cell is stimulated with or more proinflammatory cytokines to upregulate PTX3 expression. In some embodiments, the proinflammatory cytokine is IL-1 or TNF- ⁇ .
  • PTX3 for use in the methods is a native PTX3 protein isolated from a cell.
  • the cell is stimulated with or more proinflammatory cytokines to upregulate PTX3 expression.
  • the proinflammatory cytokine is IL-1 or TNF- ⁇ .
  • PTX3 is prepared by recombinant technology. In some embodiments, PTX3 is expressed from a recombinant expression vector. In some embodiments, nucleic acid encoding PTX3 is operably linked to a constitutive promoter. In some embodiments, nucleic acid encoding PTX3 is operably linked to an inducible promoter. In some embodiments, PTX3 is expressed in a transgenic animal. In some embodiments, PTX3 is a recombinant protein. In some embodiments, PTX3 is a recombinant protein isolated from a cell. In some embodiments, PTX3 is a recombinant protein produced in a cell-free extract.
  • PTX3 is purified from amniotic membrane, umbilical cord, umbilical cord amniotic membrane, chorionic membrane, amniotic fluid, or a combination thereof. In some embodiments, PTX3 is purified from amniotic membrane cells. In some embodiments, the amniotic membrane cell is an amniotic epithelial cell. In some embodiments, the amniotic membrane cell is an umbilical cord epithelial cell. In some embodiments, the amniotic membrane cell is an amniotic stromal cell. In some embodiments, the amniotic membrane cell is an umbilical cord stromal cell. In some embodiments, the amniotic membrane cell is stimulated with or more proinflammatory cytokines to upregulate PTX3 expression. In some embodiments, the proinflammatory cytokine is IL-1 or TNF- ⁇ .
  • PTX3 is not isolated from a cell or a plurality of cells (e.g., a tissue extract).
  • PTX3 comprises a fragment of PTX3 sufficient to facilitate the formation of rcHC-HA/PTX3 complex.
  • Variants of PTX3 for use in the provided methods include variants with an amino acid modification that is an amino acid replacement (substitution), deletion or insertion. In some embodiments, such modification improves one or more properties of the PTX3 polypeptides such as improving the one or more therapeutic properties of the rcHC-HA/PTX3 complex (e.g., anti-inflammatory, anti-immune, anti-angiogenic, anti-scarring, anti-adhesion, regeneration or other therapeutic activities as described herein).
  • PTX3 protein is obtained from a commercial source.
  • An exemplary commercial source for PTX3 is, but is not limited to, PTX3 (Catalog No. 1826-TS; R&D Systems, Minneapolis, Minn.).
  • the PTX3 protein used in the methods is a multimeric protein. In some embodiments, the PTX3 protein used in the methods is a homomultimer. In some embodiments, the homomultimer is a dimer, trimer, tetramer, hexamer, pentamer, or octamer. In some embodiments, the PTX3 homomultimer is a trimer, tetramer, or octamer. In particular embodiments, the PTX3 homomultimer is an octamer. In some embodiments, the multimerization domain is modified to improve multimerization of the PTX3 protein. In some embodiments, the multimerization domain is replaced with a heterogeneous multimerization domain (e.g., an Fc multimerization domain or leucine zipper) that when fused to PTX3 improves the multimerization of PTX3.
  • a heterogeneous multimerization domain e.g., an Fc multimerization domain or leucine zipper
  • TSG-6 for use in the methods is isolated from a cell or a plurality of cells (e.g., a tissue extract).
  • Exemplary cells suitable for the expression of TSG-6 include, but are not limited to, animal cells including, but not limited to, mammalian cells, primate cells, human cells, rodent cells, insect cells, bacteria, and yeast, and plant cells, including, but not limited to, algae, angiosperms, gymnosperms, pteridophytes and bryophytes.
  • TSG-6 for use in the methods is isolated from a human cell.
  • TSG-6 for use in the methods is isolated from a cell that is stimulated with one or more proinflammatory cytokines to upregulate TSG-6 expression.
  • the proinflammatory cytokine is IL-1 or TNF- ⁇ .
  • TSG-6 for use in the methods is isolated from an amniotic membrane cell. In some embodiments, TSG-6 for use in the methods is isolated from an amniotic membrane cell from an umbilical cord. In some embodiments, TSG-6 for use in the methods is isolated from an amniotic membrane cell that is stimulated with one or more proinflammatory cytokines to upregulate TSG-6 expression. In some embodiments, the proinflammatory cytokine is IL-1 or TNF- ⁇ .
  • TSG-6 for use in the methods is isolated from an umbilical cord cell. In some embodiments, TSG-6 for use in the methods is isolated from an umbilical cord cell that is stimulated with one or more proinflammatory cytokines to upregulate TSG-6 expression. In some embodiments, the proinflammatory cytokine is IL-1 or TNF- ⁇ .
  • TSG-6 for use in the methods is isolated from an amniotic epithelial cell. In some embodiments, TSG-6 for use in the methods is isolated from an umbilical cord epithelial cell. In some embodiments, TSG-6 for use in the methods is isolated from an amniotic epithelial cell or an umbilical cord epithelial cell that is stimulated with one or more proinflammatory cytokines to upregulate TSG-6 expression. In some embodiments, the proinflammatory cytokine is IL-1 or TNF- ⁇ .
  • TSG-6 for use in the methods is isolated from an amniotic stromal cell. In some embodiments TSG-6 for use in the methods is isolated from an umbilical cord stromal cell. In some embodiments, TSG-6 for use in the methods is isolated from an amniotic stromal cell or an umbilical cord stromal cell that is stimulated with one or more proinflammatory cytokines to upregulate TSG-6 expression. In some embodiments, the proinflammatory cytokine is IL-1 or TNF- ⁇ .
  • TSG-6 for use in the methods is a native TSG-6 protein isolated from a cell.
  • the cell is stimulated with or more proinflammatory cytokines to upregulate TSG-6 expression.
  • the proinflammatory cytokine is IL-1 or TNF- ⁇ .
  • TSG-6 is prepared by recombinant technology. In some embodiments, TSG-6 is expressed from a recombinant expression vector. In some embodiments, nucleic acid encoding TSG-6 is operably linked to a constitutive promoter. In some embodiments, nucleic acid encoding TSG-6 is operably linked to an inducible promoter. In some embodiments, TSG-6 is expressed in a transgenic animal. In some embodiments, TSG-6 is a recombinant protein. In some embodiments, TSG-6 is a recombinant protein isolated from a cell. In some embodiments, TSG-6 is a recombinant protein produced in a cell-free extract.
  • TSG-6 is purified from amniotic membrane, amniotic membrane, chorionic membrane, amniotic fluid, or a combination thereof. In some embodiments, TSG-6 is purified from amniotic membrane cells. In some embodiments, the amniotic membrane cell is an amniotic epithelial cell. In some embodiments, the amniotic epithelial cell is an umbilical cord epithelial cell. In some embodiments, the amniotic membrane cell is an amniotic stromal cell. In some embodiments, the amniotic membrane cell is an umbilical cord stromal cell. In some embodiments, the amniotic membrane cell is stimulated with or more proinflammatory cytokines to upregulate TSG-6 expression. In some embodiments, the proinflammatory cytokine is IL-1 or TNF- ⁇ .
  • TSG-6 is not isolated from a cell or a plurality of cells (e.g., a tissue extract).
  • TSG-6 comprises a fragment of TSG-6 that is sufficient to facilitate or catalyze the transfer HC1 of I ⁇ I to HA. In some embodiments, TSG-6 comprises the link module of TSG-6.
  • TSG-6 comprises an affinity tag.
  • affinity tags include but are not limited to a hemagglutinin tag, a poly-histidine tag, a myc tag, a FLAG tag, a glutathione-S-transferase (GST) tag.
  • GST glutathione-S-transferase
  • Such affinity tags are well known in the art for use in purification.
  • such an affinity tag incorporated into the TSG-6 polypeptide as a fusion protein or via a chemical linker.
  • TSG-6 comprises an affinity tag and the unbound TSG-6 is removed from the rcHC-HA/PTX3 complex by affinity purification.
  • TSG-6 protein is obtained from a commercial source.
  • An exemplary commercial source for TSG-6 is, but is not limited to, TSG-6 (Catalog No. 2104-TS R&D Systems, Minneapolis, Minn.).
  • the I ⁇ I comprises an HC1 chain. In some embodiments, the I ⁇ I comprises an HC1 and an HC2 chain. In some embodiments, the I ⁇ I comprises an HC1, and HC2 chain and bikunin. In some embodiments, the I ⁇ I comprises an HC1, and HC2 chain and bikunin linked by a chondroitin sulfate chain.
  • I ⁇ I is isolated from a biological sample.
  • the biological sample is a biological sample from a mammal. In some embodiments, the mammal is a human. In some embodiments, the biological sample is a blood, serum, plasma, liver, amniotic membrane, chorionic membrane or amniotic fluid sample. In some embodiments, the biological sample is a blood, serum, or plasma sample. In some embodiments, the biological sample is a blood sample. In some embodiments, the biological sample is a serum sample. In some embodiments, the biological sample is a plasma sample. In some embodiments, the I ⁇ I is purified from human blood, plasma or serum. In some embodiments, I ⁇ I is isolated from human serum.
  • I ⁇ I is not isolated from serum.
  • I ⁇ I for use in the methods is produced in an amniotic membrane cell.
  • I ⁇ I for use in the methods is produced in an umbilical cord cell.
  • I ⁇ I for use in the methods is produced in an amniotic membrane cell from an umbilical cord.
  • I ⁇ I for use in the methods is produced in an amniotic epithelial cell.
  • I ⁇ I for use in the methods is produced in an umbilical cord epithelial cell.
  • I ⁇ I for use in the methods is produced in an amniotic stromal cell.
  • I ⁇ I for use in the methods is produced in an umbilical cord stromal cell. In some embodiments, I ⁇ I for use in the methods is produced in a hepatic cell. In some embodiments, I ⁇ I is prepared by recombinant technology.
  • HC1 of I ⁇ I is isolated from a biological sample.
  • the biological sample is a biological sample from a mammal. In some embodiments, the mammal is a human. In some embodiments, the biological sample is a blood, serum, plasma, liver, amniotic membrane, chorionic membrane or amniotic fluid sample. In some embodiments, the biological sample is a blood, serum, or plasma sample. In some embodiments, the biological sample is a blood sample. In some embodiments, the biological sample is a serum sample. In some embodiments, the biological sample is a plasma sample. In some embodiments, the HC1 of I ⁇ I is purified from human blood, plasma or serum. In some embodiments, I ⁇ I is isolated from human serum.
  • HC1 of I ⁇ I is not purified from serum. In some embodiments, HC1 of I ⁇ I is prepared by recombinant technology. In some embodiments, HC1 of I ⁇ I is purified from hepatic cells. In some embodiments, HC1 of I ⁇ I is purified from amniotic membrane cells. In some embodiments, HC1 of I ⁇ I is purified from amniotic epithelial cells or umbilical cord epithelial cells. In some embodiments, HC1 of I ⁇ I is purified from amniotic stromal cells or umbilical cord stromal cells.
  • HC2 of I ⁇ I is isolated from a biological sample.
  • the biological sample is a biological sample from a mammal. In some embodiments, the mammal is a human. In some embodiments, the biological sample is a blood, serum, plasma, liver, amniotic membrane, chorionic membrane or amniotic fluid sample. In some embodiments, the biological sample is a blood, serum, or plasma sample. In some embodiments, the biological sample is a blood sample. In some embodiments, the biological sample is a serum sample. In some embodiments, the biological sample is a plasma sample. In some embodiments, the HC2 of I ⁇ I is purified from human blood, plasma or serum.
  • HC2 of I ⁇ I is isolated from human serum. In some embodiments, HC2 of I ⁇ I is isolated from human serum. In some embodiments, HC2 of I ⁇ I is not isolated from blood serum. In some embodiments, HC2 of I ⁇ I is prepared by recombinant technology. In some embodiments, HC2 of I ⁇ I is purified from hepatic cells. In some embodiments, HC2 of I ⁇ I is purified from amniotic membrane cells. In some embodiments, HC2 of I ⁇ I is purified from amniotic epithelial cells or umbilical cord epithelial cells. In some embodiments, HC2 of I ⁇ I is purified from amniotic stromal cells or umbilical cord stromal cells.
  • HA is purified from a cell, tissue or a fluid sample.
  • HA is obtained from a commercial supplier (e.g., Sigma Aldrich or Advanced Medical Optics, Irvine, Calif. (e.g., Healon)).
  • HA is obtained from a commercial supplier as a powder.
  • HA is expressed in a cell.
  • Exemplary cells suitable for the expression of HA include, but are not limited to, animal cells including, but not limited to, mammalian cells, primate cells, human cells, rodent cells, insect cells, bacteria, and yeast, and plant cells, including, but not limited to, algae, angiosperms, gymnosperms, pteridophytes and bryophytes.
  • HA is expressed in a human cell. In some embodiments, HA is expressed in a transgenic animal. In some embodiments, HA is obtained from a cell that expresses a hyaluronan synthase (e.g., HAS1, HAS2, and HAS3). In some embodiments, the cell contains a recombinant expression vector that expresses an HA synthase. In certain instances, an HA synthase lengthens hyaluronan by repeatedly adding glucuronic acid and N-acetylglucosamine to the nascent polysaccharide as it is extruded through the cell membrane into the extracellular space.
  • a hyaluronan synthase e.g., HAS1, HAS2, and HAS3
  • the cell contains a recombinant expression vector that expresses an HA synthase.
  • an HA synthase lengthens hyaluronan by repeatedly adding glucuronic
  • the weight average molecular weight of HMW HA is greater than about 500 kilodaltons (kDa), such as, for example, between about 500 kDa and about 10,000 kDa, between about 800 kDa and about 8,500 kDa, between about 1100 kDa and about 5,000 kDa, or between about 1400 kDa and about 3,500 kDa. In some embodiments, the weight average molecular weight of HMW HA is about 3000 kDa.
  • one or more additional components are added to generate an rcHC-HA/PTX3 complex.
  • a small leucine rich proteoglycan (SLRP) is added to generate an rcHC-HA/PTX3 complex.
  • the SLRP is a class I, class II or class II SLRP.
  • the SLRP is selected from among class I SLRPs, such as decorin and biglycan.
  • the SLRP is selected from among class II SLRPs, such as fibromodulin, lumican, PRELP (proline arginine rich end leucine-rich protein), keratocan, and osteoadherin.
  • the SLRP is selected from among class III SLRPs, such as epipycan and osteoglycin. In some embodiments, the SLRP is selected from among bikunin, decorin, biglycan, and osteoadherin. In some embodiments, the SLRP comprises a glycosaminoglycan. In some embodiments, the SLRP comprises keratan sulfate.
  • HMW HA is immobilized by any suitable method.
  • HMW HA is immobilized to a solid support, such as culture dish, bead, a column or other suitable surfaces, such as, for example, a surface of an implantable medical device or a portion thereof or on a surface that is subsequently connected to or combined with an implantable medical device as described herein.
  • HMW HA is immobilized directly to the solid support, such as by chemical linkage.
  • HMW HA is attached indirectly to the solid support via a linker or an intermediary protein.
  • HMW HA is immobilized directly to the solid support via crosslinking to the solid support. In some embodiments, HMW HA is immobilized directly to the solid support without crosslinking to the solid support. In some embodiments, HMW HA is immobilized directly to the solid support as a coating. In some embodiments, HMW HA is immobilized to a CovalinkTM-NH surface. In some embodiments, HMW HA is immobilized directly to the solid support as a coating. In some embodiments, HMW HA is immobilized to a CovalinkTM-NH surface for about 16 h at 4° C.
  • the method comprises immobilizing HMW HA to a solid surface via direct linkage to a solid support (i.e. without an intermediary protein).
  • the solid support is washed to remove unbound HMW HA prior to contacting the immobilized HA with I ⁇ I, TSG-6, and PTX3.
  • the solid support is washed with washes of 8 M GnHCl and PBS to remove unbound HMW HA prior to contacting the immobilized HA with I ⁇ I, TSG-6, and PTX3.
  • the method comprises immobilizing HA to a solid surface via an intermediary protein or a linker.
  • the linker is a peptide linker.
  • the intermediary protein is an HA binding protein (HABP).
  • HABP is first attached to a solid support (e.g., by cross-linking, chemical linkage or via a chemical linker).
  • the solid support comprising HABP is then contacted with HA (e.g., HMW HA) to immobilize HA to the solid support via binding of the HABP to HA.
  • the solid support is washed to remove unbound HMW HA prior to contacting the immobilized HMW HA with I ⁇ I, TSG-6, and PTX3. In some embodiments, the solid support is washed with washes of 8 M GnHCl and PBS to remove unbound HMW HA prior to contacting the immobilized HA with I ⁇ I, TSG-6, and PTX3.
  • the method comprises immobilizing HA to a solid surface via attachment of a peptide linker to the solid support and attachment HA to the peptide linker.
  • the peptide linker comprises a protease cleavage site.
  • the method comprises immobilizing HA to a solid surface via attachment of a cleavable chemical linker, such as, but not limited to a disulfide chemical linker.
  • a cleavable chemical linker such as, but not limited to a disulfide chemical linker.
  • the HABP selected for use in the methods is an HABP that is dissociated from HA following formation of the rcHC-HA/PTX3 complex.
  • the HABP non-covalently binds to HA.
  • the method further comprises dissociating the rcHC-HA/PTX3 complex from HABP using one or more dissociating agents.
  • Dissociating agents for the disruption of non-covalent interactions e.g., guanidine hydrochloride, urea and various detergents, e.g., SDS
  • the dissociating agent is urea.
  • the dissociating agent is guanidine hydrochloride.
  • the dissociation agent is about 4M to about 8M guanidine-HCl. In some embodiments, the dissociation agent is about 4M, about 5M, about 6M, about 7M, about 8M guanidine-HCl. In some embodiments, the dissociation agent is about 4M to about 8M guanidine-HCl in PBS at pH 7.5.
  • such dissociating agents are employed to dissociate the rcHC-HA/PTX3 complex from an intermediary HABP.
  • An HABP for use in the methods typically is selected such that the binding affinity for HA is strong enough to permit assembly of the rcHC-HA/PTX3 complex but is dissociated from the rcHC-HA/PTX3 complex with a suitable dissociation agent.
  • the dissociating agent is guanidine hydrochloride.
  • Exemplary HABPs for use with the methods provided herein include, but are not limited to, HAPLN1, HAPLN2, HAPLN3, HAPLN4, aggrecan, versican, neurocan, brevican, phosphacan, TSG-6, CD44, stabilin-1, stabilin-2, or portions thereof (e.g., link modules thereof) sufficient to bind HA.
  • the HABP is versican.
  • the HABP is a recombinant protein.
  • the HABP is a recombinant mammalian protein.
  • the HABP is a recombinant human protein.
  • the HABP is a recombinant versican protein or a portion thereof sufficient to bind to HA. In some embodiments, the HABP is a recombinant aggrecan protein or a portion thereof sufficient to bind to HA. In some embodiments, the HABP is a native HABP or a portion thereof sufficient to bind to HA. In some embodiments, the native HABP is isolated from mammalian tissue or cells. In some embodiments, the HABP is isolated from bovine nasal cartilage (e.g. HABP from Seikagaku which contains the HA binding domains of aggrecan and link protein).
  • bovine nasal cartilage e.g. HABP from Seikagaku which contains the HA binding domains of aggrecan and link protein.
  • the HABP comprises a link module of HAPLN1, HAPLN2, HAPLN3, HAPLN4, aggrecan, versican, neurocan, brevican, phosphacan, TSG-6, CD44, stabilin-1, or stabilin-2.
  • the HABP comprises a link module of versican.
  • the HABP comprising a link module is a recombinant protein.
  • the HABP comprising a link module of versican is a recombinant protein.
  • an intermediary protein such as an HABP
  • a site-specific protease such as furin, 3C protease, caspase, matrix metalloproteinase or TEV protease.
  • assembled rcHC-HA/PTX3 complexes are released from the solid support by contacting the immobilized complexes with a protease that cleaves the specific cleavage sequence.
  • the rcHC-HA/PTX3 complex is purified. In some embodiments, the rcHC-HA/PTX3 complex is purified by any suitable method or combination of methods. The embodiments described below are not intended to be exclusive, only exemplary.
  • the rcHC-HA/PTX3 complex is purified by chromatography (e.g., ion exchange, affinity, size exclusion, and hydroxyapatite chromatography), tangential flow filtration (TFF), gel filtration, centrifugation (e.g., gradient centrifugation), or differential solubility, ethanol precipitation or by any other available technique for the purification of proteins.
  • chromatography e.g., ion exchange, affinity, size exclusion, and hydroxyapatite chromatography
  • TMF tangential flow filtration
  • gel filtration e.g., gel filtration
  • centrifugation e.g., gradient centrifugation
  • differential solubility ethanol precipitation or by any other available technique for the purification of proteins.
  • the rcHC-HA/PTX3 complex is purified by immunoaffinity chromatography.
  • antibodies are generated against a component of the rcHC-HA/PTX3 complex (e.g., anti-HC1, anti-PTX3, an antibody against one or more SLRPs of the rcHC-HA/PTX3 complex, e.g., anti-bikunin, anti-decorin, anti-biglycan, or anti-osteoadherin) and affixed to a solid support.
  • the unpurified rcHC-HA/PTX3 complex i.e., the mobile phase
  • the rcHC-HA/PTX3 complex binds to the antibodies.
  • the support is washed (e.g., with PBS) to remove any unbound or loosely bound molecules.
  • the support is then washed with a solution that enables elution of the rcHC-HA/PTX3 complex from the support (e.g., 1% SDS, 6M guanidine-HCl, or 8M urea).
  • the dissociating agent is removed from the dissociated rcHC-HA/PTX3 complex.
  • the dissociating agent is removed from the dissociated rcHC-HA/PTX3 complex by a method including, but not limited to, ion-exchange chromatography, dialysis, tangential flow filtration (TFF), gel filtration chromatography, ultrafiltration, or diafiltration.
  • a method including, but not limited to, ion-exchange chromatography, dialysis, tangential flow filtration (TFF), gel filtration chromatography, ultrafiltration, or diafiltration.
  • the rcHC-HA/PTX3 complex is purified by affinity chromatography.
  • an HABP is employed to bind to the rcHC-HA/PTX3 complex for purification of the complex and affixed to a stationary support.
  • the unpurified rcHC-HA/PTX3 complex i.e., the mobile phase
  • the rcHC-HA/PTX3 complex binds to the HABP.
  • the support is washed (e.g., with PBS) to remove any unbound or loosely bound molecules.
  • the support is then washed with a solution (e.g., a dissociating agent) that enables elution of the rcHC-HA/PTX3 complex from the support.
  • a solution e.g., a dissociating agent
  • the dissociating agent is removed from the dissociated rcHC-HA/PTX3 complex by a method including, but not limited to, ion-exchange chromatography, dialysis, tangential flow filtration (TFF), gel filtration chromatography, ultrafiltration, or diafiltration.
  • the rcHC-HA/PTX3 complex is purified by a combination of HABP affinity chromatography, and immunoaffinity chromatography using antibodies against one or more components of the rcHC-HA/PTX3 complex.
  • one or more components of the rcHC-HA/PTX3 complex disclosed herein comprise an affinity tag (e.g., a fusion protein of PTX3 or HC1 with an affinity tag).
  • affinity tags that are incorporated into one or more components of the rcHC-HA/PTX3 complex in some embodiments include, but are not limited to, a hemagglutinin tag, poly-histidine, a myc tag, a FLAG tag, or glutathione-S-transferase sequence.
  • the ligand for the affinity tag is affixed to the solid support.
  • the unpurified rcHC-HA/PTX3 complex is passed over the support.
  • the rcHC-HA/PTX3 complex binds to the ligand.
  • the support is washed (e.g., with PBS) to remove any unbound or loosely bound molecules.
  • the support is then washed with a solution that enables elution of an rcHC-HA/PTX3 complex disclosed herein from the support.
  • the elution agent is removed from the dissociated rcHC-HA/PTX3 complex by a method including, but not limited to, ion-exchange chromatography, dialysis, tangential flow filtration (TFF), gel filtration chromatography, ultrafiltration, or diafiltration.
  • the PTX3, TSG-6, and/or HC1 are conjugated to a label.
  • a “label” refers to a detectable compound or composition which is conjugated directly or indirectly to a polypeptide so as to generate a labeled polypeptide.
  • the label is detectable by itself (e.g., radioisotope labels or fluorescent labels) or, in the case of an enzymatic label, catalyzes chemical alteration of a substrate compound composition which is detectable.
  • labels include fluorogenic moieties, dyes, fluorescent tags, green fluorescent protein, or luciferase.
  • nHC-HA/PTX3 and rcHC-HA/PTX3 complexes are assessed by any suitable method including, in vitro and in vivo methods.
  • Exemplary in vitro methods are provided herein and include, but are not limited, to cell culture methods that assess the ability of nHC-HA/PTX3 or rcHC-HA/PTX3 complexes to promote attachment of macrophages to the immobilized nHC-HA/PTX3 or rcHC-HA/PTX3 complexes, to inhibit or reduce aggregation of macrophages, to promote apoptosis of neutrophils, macrophage phagocytosis of apoptotic neutrophils, and M2 polarization of stimulated macrophages.
  • the macrophages used in the assay are stimulated, such as by exposure to LPS or IFN- ⁇ .
  • the gene or protein expression in stimulated macrophages is assessed following contact with nHC-HA/PTX3 or rcHC-HA/PTX3 complexes.
  • a suitable control is employed for comparison.
  • the control is the absence of treatment with an nHC-HA/PTX3 or rcHC-HA/PTX3 complex (i.e. a negative control).
  • the activity of an rcHC-HA/PTX3 complex is compared to the activity of a native HC-HA/PTX3 complex.
  • the native HC-HA/PTX3 is isolated from amniotic membrane.
  • gene expression in treated macrophages is assessed by PCR, RT-PCR, Northern blotting, western blotting, dot blotting, immunohistochemistry, chromatography or other suitable method of detecting proteins or nucleic acids.
  • the level of expression of IL-10, IL-12, IL23, LIGHT and SPHK1 is assessed.
  • compositions comprising nHC-HA/PTX3 or rcHC-HA/PTX3 complexes described herein.
  • pharmaceutical compositions comprising nHC-HA/PTX3 or rcHC-HA/PTX3 complexes produced by the methods provided herein.
  • the pharmaceutical compositions are formulated in a conventional manner using one or more physiologically acceptable carriers including excipients and auxiliaries which facilitate processing of an nHC-HA/PTX3 or rcHC-HA/PTX3 complex into preparations which are suitable for pharmaceutical use. Proper formulation is dependent upon the route of administration selected. Any of the well-known techniques, carriers, and excipients can be used as suitable and as understood in the art.
  • a pharmaceutical composition comprising an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein.
  • the pharmaceutical composition further comprises at least one pharmaceutically acceptable carrier.
  • the pharmaceutical composition further comprises an adjuvant, excipient, preservative, agent for delaying absorption, filler, binder, adsorbent, buffer, and/or solubilizing agent.
  • compositions that are formulated to contain an nHC-HA/PTX3 or rcHC-HA/PTX3 complex provided herein include, but are not limited to, a solution, suspension, emulsion, syrup, granule, powder, ointment, tablet, capsule, pill, tincture, transdermal system, ointment, lotion, cream, paste, foam, gel, or an aerosol.
  • an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is administered as an aqueous suspension.
  • an aqueous suspension comprises water, Ringer's solution and/or isotonic sodium chloride solution.
  • an aqueous suspension comprises a sweetening or flavoring agent, coloring matters or dyes and, if desired, emulsifying agents or suspending agents, together with diluents water, ethanol, propylene glycol, glycerin, or combinations thereof.
  • an aqueous suspension comprises a suspending agent.
  • an aqueous suspension comprises sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethyl-cellulose, sodium alginate, polyvinyl-pyrrolidone, gum tragacanth and/or gum acacia. In some embodiments, an aqueous suspension comprises a dispersing or wetting agent.
  • an aqueous suspension comprises a naturally-occurring phosphatide, for example lecithin, or condensation products of an alkylene oxide with fatty acids, for example polyoxyethylene stearate, or condensation products of ethylene oxide with long chain aliphatic alcohols, for example heptadecaethylene-oxycetanol, or condensation products of ethylene oxide with partial esters derived from fatty acids and a hexitol such as polyoxyethylene sorbitol monooleate, or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides, for example polyethylene sorbitan monooleate.
  • an aqueous suspension comprises a preservative.
  • an aqueous suspension comprises ethyl, or n-propyl p-hydroxybenzoate. In some embodiments, an aqueous suspension comprises a sweetening agent. In some embodiments, an aqueous suspension comprises sucrose, saccharin or aspartame.
  • an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is administered as an oily suspension.
  • an oily suspension is formulated by suspending the active ingredient in a vegetable oil (e.g., arachis oil, olive oil, sesame oil or coconut oil), or in mineral oil (e.g., liquid paraffin).
  • an oily suspension comprises a thickening agent (e.g., beeswax, hard paraffin or cetyl alcohol).
  • an oily suspension comprises sweetening agents (e.g., those set forth above).
  • an oily suspension comprises an anti-oxidant (e.g., butylated hydroxyanisol or alpha-tocopherol).
  • an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is formulated for parenteral injection (e.g., via injection or infusion, including intraarterial, intracardiac, intradermal, intraduodenal, intramedullary, intramuscular, intraosseous, intraperitoneal, intrathecal, intravascular, intravenous, intravitreal, epidural and/or subcutaneous).
  • an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is administered as a sterile solution, suspension or emulsion.
  • a formulation for parenteral administration includes aqueous and/or non-aqueous (oily) sterile injection solutions of an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein, which in some embodiments, contain antioxidants, buffers, bacteriostats and/or solutes which render the formulation isotonic with the blood of the intended recipient; and/or aqueous and/or non-aqueous sterile suspensions which in some embodiments, include a suspending agent and/or a thickening agent.
  • a formulation for parenteral administration includes suitable stabilizers or agents which increase the solubility of an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein to allow for the preparation of highly concentrated solutions.
  • an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is administered as an oil-in-water micro-emulsion where the active ingredient is dissolved in the oily phase.
  • an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is dissolved in a fatty oil (e.g., sesame oil, or synthetic fatty acid esters, (e.g., ethyl oleate or triglycerides, or liposomes.
  • a fatty oil e.g., sesame oil, or synthetic fatty acid esters, (e.g., ethyl oleate or triglycerides, or liposomes.
  • an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is dissolved in a mixture of soybean oil and/or lecithin.
  • the oil solution is introduced into a water and glycerol mixture
  • a composition formulated for parenteral administration is administered as a single bolus shot. In some embodiments, a composition formulated for parenteral administration is administered via a continuous intravenous delivery device (e.g., Deltec CADD-PLUSTM model 5400 intravenous pump).
  • a continuous intravenous delivery device e.g., Deltec CADD-PLUSTM model 5400 intravenous pump.
  • a formulation for injection is presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative.
  • a formulation for injection is stored in powder form or in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example, saline or sterile pyrogen-free water, immediately prior to use.
  • Topical formulations include, but are not limited to, ointments, creams, lotions, solutions, pastes, gels, films, sticks, liposomes, nanoparticles.
  • a topical formulation is administered by use of a patch, bandage or wound dressing.
  • an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is formulated as composition is in the form of a solid, a cross-linked gel, or a liposome.
  • an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is formulated as an insoluble cross-linked hydrogel.
  • a topical formulation comprises a gelling (or thickening) agent.
  • Suitable gelling agents include, but are not limited to, celluloses, cellulose derivatives, cellulose ethers (e.g., carboxymethylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxymethylcellulose, hydroxypropylmethylcellulose, hydroxypropylcellulose, methylcellulose), guar gum, xanthan gum, locust bean gum, alginates (e.g., alginic acid), silicates, starch, tragacanth, carboxyvinyl polymers, carrageenan, paraffin, petrolatum, acacia (gum arabic), agar, aluminum magnesium silicate, sodium alginate, sodium stearate, bladderwrack, bentonite, carbomer, carrageenan, carbopol, xanthan, cellulose, microcrystalline cellulose (MCC), ceratonia, chondrus, dextrose, furcellaran, gelatin,
  • PEG 200-4500 gum tragacanth, ethyl cellulose, ethylhydroxyethyl cellulose, ethylmethyl cellulose, methyl cellulose, hydroxyethyl cellulose, hydroxyethylmethyl cellulose, hydroxypropyl cellulose, poly(hydroxyethyl methacrylate), oxypolygelatin, pectin, polygeline, povidone, propylene carbonate, methyl vinyl ether/maleic anhydride copolymer (PVM/MA), poly(methoxyethyl methacrylate), poly(methoxyethoxyethyl methacrylate), hydroxypropyl cellulose, hydroxypropylmethyl-cellulose (HPMC), sodium carboxymethyl-cellulose (CMC), silicon dioxide, polyvinylpyrrolidone (PVP: povidone), or combinations thereof.
  • PVM/MA methyl vinyl ether/maleic anhydride copolymer
  • HPMC sodium carboxymethyl-cellulose
  • CMC silicon
  • a topical formulation disclosed herein comprises an emollient.
  • Emollients include, but are not limited to, castor oil esters, cocoa butter esters, safflower oil esters, cottonseed oil esters, corn oil esters, olive oil esters, cod liver oil esters, almond oil esters, avocado oil esters, palm oil esters, sesame oil esters, squalene esters, kukui oil esters, soybean oil esters, acetylated monoglycerides, ethoxylated glyceryl monostearate, hexyl laurate, isohexyl laurate, isohexyl palmitate, isopropyl palmitate, methyl palmitate, decyloleate, isodecyl oleate, hexadecyl stearate decyl stearate, isopropyl isostearate, methyl isostearate, diisopropyl adip
  • an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is formulated with one or more natural polymers.
  • an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is formulated with a natural polymer that is fibronectin, collagen, laminin, keratin, fibrin, fibrinogen, hyaluronic acid, heparan sulfate, chondroitin sulfate.
  • an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is formulated with a polymer gel formulated from a natural polymer.
  • an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is formulated with a polymer gel formulated from a natural polymer, such as, but not limited to, fibronectin, collagen, laminin, keratin, fibrin, fibrinogen, hyaluronic acid, heparan sulfate, chondroitin sulfate, and combinations thereof.
  • a polymer gel formulated from a natural polymer, such as, but not limited to, fibronectin, collagen, laminin, keratin, fibrin, fibrinogen, hyaluronic acid, heparan sulfate, chondroitin sulfate, and combinations thereof.
  • an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is formulated with a cross-linked polymer.
  • an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is formulated with a non-cross-linked polymer. In some embodiments, an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is formulated with a non-cross-linked polymer and a cross-linked polymer. In some embodiments, an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is formulated with cross-linked hyaluronan gel. In some embodiments, an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is formulated with an insoluble cross-linked HA hydrogel.
  • an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is formulated with non-cross-linked hyaluronan gel.
  • an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is formulated with a collagen matrix.
  • an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is formulated with a fibrin matrix.
  • an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is formulated with a fibrin/collagen matrix.
  • an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is formulated for administration to a tumor or a tissue related thereto.
  • Formulations suitable for administration to a tumor include, but are not limited to, solutions, suspensions (e.g., an aqueous suspension), ointments, gels, creams, liposomes, niosomes, pharmacosomes, nanoparticles, or combinations thereof.
  • an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein for injection into a solid tumor is administered by injection into a tumor, the surrounding tissue, or a combination thereof.
  • an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is administered concurrent to excision of cancer cells or a tumor. In some embodiments, an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is administered to surgical margins from the excision of cancer cells or a tumor. In some embodiments, an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is administered
  • a “depot preparation” is a controlled-release formulation that is implanted in a tumor or a tissue related thereto (e.g., a surgical margin) (for example subcutaneously, intramuscularly, intravitreally, or within the subconjunctiva).
  • a depot preparation is formulated by forming microencapsulated matrices (also known as microencapsulated matrices) of an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein in biodegradable polymers.
  • a depot preparation is formulated by entrapping an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein in liposomes or microemulsions.
  • an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is formulated for rectal or vaginal administration.
  • an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is administered as a suppository.
  • a composition suitable for rectal administration is prepared by mixing an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein with a suitable non-irritating excipient which is solid at ordinary temperatures but liquid at the rectal temperature and will therefore melt in the rectum to release the drug.
  • a composition suitable for rectal administration is prepared by mixing an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein with cocoa butter, glycerinated gelatin, hydrogenated vegetable oils, mixtures of polyethylene glycols of various molecular weights or fatty acid esters of polyethylene glycol.
  • an nHC-HA/PTX3 or rcHC-HA/PTX3 complex described herein is optionally incorporated within controlled release particles, lipid complexes, liposomes, nanoparticles, microspheres, microparticles, nanocapsules or other agents which enhance or facilitate localized delivery to the skin.
  • An example of a conventional microencapsulation process for pharmaceutical preparations is described in U.S. Pat. No. 3,737,337, incorporated herein by reference for such disclosure.
  • the amount of pharmaceutical compositions administered is dependent in part on the individual being treated.
  • the daily dosage will normally be determined by the prescribing physician with the dosage generally varying according to the age, sex, diet, weight, general health and response of the individual, the severity of the individual's symptoms, the precise disease or condition being treated, the severity of the disease or condition being treated, time of administration, route of administration, the disposition of the composition, rate of excretion, drug combination, and the discretion of the prescribing physician.
  • an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is administered by local injection, directly into a tumor and/or the surrounding tissue.
  • the tumor cannot be removed surgically, or is “inoperable”.
  • an inoperable tumor is not accessible or the patient has medical conditions that limit the ability to withstand surgery.
  • a tumor is in a sensitive location, such as the spinal cord, the brain, or other tissues, where surgical removal could critically damage surrounding tissue.
  • a tumor infiltrates or invades surrounding tissue, for example with certain brain cancers, and are impossible to surgically extract without harming the surrounding tissue.
  • Inoperable tumors can arise from, without limitation, central nervous system (CNS) cancer, such as glioblastoma multiforme, breast cancer, pancreatic cancer, or bladder cancer.
  • CNS central nervous system
  • a cancer has multiple secondary tumors, or metastases, elsewhere in the body. The number of secondary tumors may be too great to remove safely.
  • Types of metastatic cancer include, without limitation, bladder cancer, breast cancer, colon cancer, kidney cancer, lung cancer, melanoma, ovarian cancer, pancreatic cancer, prostate cancer, rectal cancer, stomach cancer, thyroid cancer, liver cancer, or uterine cancer.
  • the administered dosage of an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is between about 0.001 to about 1000 mg. In some embodiments, the amount of nHC-HA/PTX3 or rcHC-HA/PTX3 complex administered is in the range of about 0.5 to about 50 mg. In some embodiments, the amount of nHC-HA/PTX3 or rcHC-HA/PTX3 complex administered is about 0.001 to about 7 g. In some embodiments, the amount of nHC-HA/PTX3 or rcHC-HA/PTX3 complex administered is about 0.01 to about 7 g.
  • the amount of nHC-HA/PTX3 or rcHC-HA/PTX3 complex administered is about 0.02 to about 5 g. In some embodiments, the amount of nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is about 0.05 to about 2.5 g. In some embodiments, the amount of nHC-HA/PTX3 or rcHC-HA/PTX3 administered is about 0.1 to about 1 g.
  • an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is administered, before, during or after the occurrence of a disease or condition.
  • a combination therapy is administered before, during or after the occurrence of a disease or condition.
  • an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is administered with a combination therapy before, during or after the occurrence of a disease or condition.
  • the timing of administering the composition containing an nHC-HA/PTX3 or rcHC-HA/PTX3 disclosed herein varies.
  • an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is used as a prophylactic and is administered continuously to subjects with a propensity to develop conditions or diseases in order to prevent the occurrence of the disease or condition.
  • an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is administered to a subject during or as soon as possible after the onset of the symptoms.
  • the administration of an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is initiated within the first 48 hours of the onset of the symptoms, preferably within the first 48 hours of the onset of the symptoms, more preferably within the first 6 hours of the onset of the symptoms, and most preferably within 3 hours of the onset of the symptoms.
  • the initial administration is via any route practical, such as, for example, an intravenous injection, a bolus injection, infusion over 5 minutes to about 5 hours, a pill, a capsule, transdermal patch, buccal delivery, or combination thereof.
  • An nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is preferably administered as soon as is practicable after the onset of a disease or condition is detected or suspected, and for a length of time necessary for the treatment of the disease, such as, for example, from about 1 month to about 3 months.
  • the length of treatment varies for each subject, and the length is determined using the known criteria.
  • an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein or a formulation containing a complex is administered for at least 2 weeks, preferably about 1 month to about 5 years, and more preferably from about 1 month to about 3 years.
  • an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is administered in a single dose, once daily. In some embodiments, an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is administered in multiple doses, more than once per day. In some embodiments, an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is administered twice daily. In some embodiments, an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is administered three times per day. In some embodiments, an nHC-HA/PTX3 or rcHC-HA/PTX3 complex is administered four times per day. In some embodiments, an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is administered more than four times per day.
  • an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is administered in a single dose. In some embodiments, an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is administered in a single dose in conjunction with a tumor excision, cryoablation, or radiofrequency ablation.
  • an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is administered for prophylactic and/or therapeutic treatments.
  • an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is administered to an individual already suffering from a disease or condition, in an amount sufficient to cure or at least partially arrest the symptoms of the disease or condition. Amounts effective for this use will depend on the severity and course of the disease or condition, previous therapy, the individual's health status, weight, and response to the drugs, and the judgment of the treating physician.
  • an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is administered to an individual that is at risk of a particular disorder.
  • Such an amount is defined to be a “prophylactically effective amount or dose.”
  • the precise amounts also depend on the individual's state of health, weight, and other physical parameters of the individual.
  • an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is administered chronically, that is, for an extended period of time, including throughout the duration of the individual's life in order to ameliorate or otherwise control or limit the symptoms of the individual's disease or condition.
  • an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is administered continuously or the dose of drug being administered is temporarily reduced or temporarily suspended for a certain length of time (i.e., a “drug holiday”).
  • the length of the drug holiday varies between 2 days and 1 year, including by way of example only, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 12 days, 15 days, 20 days, 28 days, 35 days, 50 days, 70 days, 100 days, 120 days, 150 days, 180 days, 200 days, 250 days, 280 days, 300 days, 320 days, 350 days, or 365 days.
  • the dose reduction during a drug holiday is from 10%-100%, including, by way of example only, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%.
  • a maintenance dose is administered if necessary.
  • the dosage or the frequency of administration, or both is reduced, as a function of the symptoms, to a level at which the improved disease, disorder or condition is retained.
  • individuals require intermittent treatment on a long-term basis upon any recurrence of symptoms.
  • the pharmaceutical composition described herein is in unit dosage forms suitable for single administration of precise dosages.
  • the formulation is divided into unit doses containing appropriate quantities of an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein.
  • the unit dosage is in the form of a package containing discrete quantities of the formulation.
  • Non-limiting examples are packaged tablets or capsules, and powders in vials or ampoules.
  • aqueous suspension compositions are packaged in single-dose non-reclosable containers.
  • multiple-dose reclosable containers are used, in which case it is typical to include a preservative in the composition.
  • formulations for parenteral injection are presented in unit dosage form, which include, but are not limited to ampoules, or in multi dose containers, with an added preservative.
  • the area surrounding the tumor is contacted with at least or about 1 microgram (ug), 10 ug, 20 ug, 30 ug, 40 ug, 50 ug, 60 ug, 70 ug, 80 ug, 90 ug, 100 ug, 200 ug, 300 ug, 400 ug, 500 ug, 600 ug, 700 ug, 800 ug, 900 ug, 1000 ug, or more than 1000 ug of the HC-HA/PTX3 complex.
  • ug microgram
  • the area surrounding the tumor is contacted with at least or about 1 milligram (mg), 10 mg, 20 mg, 30 mg, 40 mg, 50 mg, 60 mg, 70 mg, 80 mg, 90 mg, 100 mg, 200 mg, 300 mg, 400 mg, 500 mg, or more than 500 mg of the HC-HA/PTX3 complex.
  • the area surrounding the tumor is contacted with a range of about 1 to 10, 1 to 20, 1 to 40, 1 to 60, 1 to 80, 1 to 100, 1 to 150, 1 to 200, 10 to 20, 10 to 40, 10 to 60, 10 to 80, 10 to 100, 10 to 150, 10 to 200, 20 to 40, 20 to 60, 20 to 80, 20 to 100, 20 to 150, 20 to 200, 40 to 60, 40 to 80, 40 to 100, 40 to 150, 40 to 200, 60 to 80, 60 to 100, 60 to 150, 60 to 200, 80 to 100, 80 to 150, 80 to 200, 100 to 150, 100 to 200, or 150 to 200 microgram (ug) of the HC-HA/PTX3 complex.
  • ug microgram
  • the area surrounding the tumor is contacted with a range of about 1 to 10, 1 to 20, 1 to 40, 1 to 60, 1 to 80, 1 to 100, 1 to 150, 1 to 200, 10 to 20, 10 to 40, 10 to 60, 10 to 80, 10 to 100, 10 to 150, 10 to 200, 20 to 40, 20 to 60, 20 to 80, 20 to 100, 20 to 150, 20 to 200, 40 to 60, 40 to 80, 40 to 100, 40 to 150, 40 to 200, 60 to 80, 60 to 100, 60 to 150, 60 to 200, 80 to 100, 80 to 150, 80 to 200, 100 to 150, 100 to 200, or 150 to 200 milligram (mg) of the HC-HA/PTX3 complex.
  • mg milligram
  • the daily dosages appropriate for an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein are, for example, from about 0.01 to 100 mg.
  • An indicated daily dosage in the larger mammal, including, but not limited to, humans, is in the range from about 10 ug to about 100 mg from about 0.5 mg to about 100 mg, conveniently administered in divided doses, including, but not limited to, up to four times a day or in extended release form.
  • Suitable unit dosage forms for oral administration include from about 1 to 50 mg active ingredient.
  • Suitable doses for injection into a tumor and/or surrounding tissues is in the range from about 0.1 to about 100 mg per injection.
  • the dosages are altered depending on a number of variables, not limited to the activity of an nHC-HA/PTX3 or rcHC-HA/PTX3 complex used, the disease or condition to be treated, the mode of administration, the requirements of the individual subject, the severity of the disease or condition being treated, and the judgment of the practitioner.
  • the toxicity and therapeutic efficacy of such therapeutic regimens are determined by standard pharmaceutical procedures in cell cultures or experimental animals, including, but not limited to, the determination of the LD 50 (the dose lethal to 50% of the population) and the ED 50 (the dose therapeutically effective in 50% of the population).
  • the dose ratio between the toxic and therapeutic effects is the therapeutic index and it is expressed as the ratio between LD 50 and ED 50 .
  • nHC-HA/PTX3 or rcHC-HA/PTX3 complexes exhibiting high therapeutic indices are preferred.
  • the data obtained from cell culture assays and animal studies is used in formulating a range of dosage for use in human.
  • the dosage of an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein lies preferably within a range of circulating concentrations that include the ED 50 with minimal toxicity. In some embodiments, the dosage varies within this range depending upon the dosage form employed and the route of administration utilized.
  • the pharmaceutical compositions of nHC-HA/PTX3 or rcHC-HA/PTX3 complexes are packaged as articles of manufacture containing packaging material, a pharmaceutical composition which is effective for prophylaxis and/or treating a disease or condition, and a label that indicates that the pharmaceutical composition is to be used for treating the disease or condition.
  • the pharmaceutical compositions are packaged in unit dosage forms contain an amount of the pharmaceutical composition for a single dose or multiple doses.
  • the packaged compositions contain a lyophilized powder of the pharmaceutical compositions, which is reconstituted (e.g., with water or saline) prior to administration.
  • an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is assembled directly on a surface of or formulated as a coating for an implantable medical device.
  • Methods for covalent attachment of hyaluronan to surfaces such as, but not limited to, metallic, polymeric, ceramic, silica and composite surfaces is well-known in the art and in some embodiments, is employed in conjunction with the methods provided herein for the assembly of nHC-HA/PTX3 or rcHC-HA/PTX3 complexes on such surfaces (see e.g., U.S. Pat. Nos.
  • an nHC-HA/PTX3 or rcHC-HA/PTX3 complex is assembled directly on a surface of an implantable medical device or a portion thereof.
  • an nHC-HA/PTX3 or rcHC-HA/PTX3 complex that has been generated according the methods provided herein is purified and then attached directly on a surface of an implantable medical device or a portion thereof.
  • an nHC-HA/PTX3 or rcHC-HA/PTX3 complex that has been generated according the methods provided herein is purified and then formulated as a coating for attachment to the medical device or a portion thereof.
  • the coating is applied directly to the surfaces or is applied to a pretreated or coated surface where the pretreatment or coating is designed to aid adhesion of the coating to the substrate.
  • an nHC-HA/PTX3 or rcHC-HA/PTX3 complex that has been generated according the methods provided herein is purified and then attached to a medical device or a portion thereof that has been coated with a substance that promotes the attachment of the nHC-HA/PTX3 or rcHC-HA/PTX3 complex.
  • the medical device or a portion thereof is coated with an adhesive polymer that provides functional groups on its surface for the covalent attachment of hyaluronan of the nHC-HA/PTX3 or rcHC-HA/PTX3 complex.
  • a coupling agent such as, but not limited to carbodiimide is employed to attach the nHC-HA/PTX3 or rcHC-HA/PTX3 complex to the polymer coating.
  • photoimmobilization is employed to covalently attach an nHC-HA/PTX3 or rcHC-HA/PTX3 complex that has been generated according the methods provided herein to medical device or a portion thereof.
  • an nHC-HA/PTX3 or rcHC-HA/PTX3 complex that has been generated according the methods provided herein is attached to a medical device or a portion thereof using a spacer molecule that comprises a photochemically or thermochemically reactive group.
  • the coating formulations comprising an nHC-HA/PTX3 or rcHC-HA/PTX3 complex are applied to the substrate by for example dip-coating.
  • Other methods of application include, but are not limited to, spray, wash, vapor deposition, brush, roller, curtain, spin coating and other methods known in the art.
  • Exemplary implantable medical devices include, but are not limited to a bone implant, wound drain, shunt, urethral insert, metal or plastic implant, stent, stent graft, vascular graft, pellets, wafers, implantable drug pump, drug delivery system, microparticle, nanoparticle, and microcapsule.
  • an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is attached to the microcapsule or assembled directly on a microcapsule.
  • the nHC-HA/PTX3 or rcHC-HA/PTX3 complex is combined with a material used to form the microcapsule and a microcapsule is generated that contains the nHC-HA/PTX3 or rcHC-HA/PTX3 complex.
  • the nHC-HA/PTX3 or rcHC-HA/PTX3 complex is used to coat the inner surface of the microcapsule.
  • the nHC-HA/PTX3 or rcHC-HA/PTX3 complex is used to coat the outer surface of the microcapsule. In some embodiments, the nHC-HA/PTX3 or rcHC-HA/PTX3 complex is used to coat the inner and outer surface of the microcapsule.
  • compositions and methods described herein are used in conjunction with a second or further or additional therapeutic agent in addition to the native or reconstituted HC-HA/PTX3 complex. In some embodiments, the compositions and methods described herein are used in conjunction with two or more therapeutic agents. In some embodiments, the compositions and methods described herein are used in conjunction with one or more therapeutic agents. In some embodiments, the compositions and methods described herein are used in conjunction with 2, 3, 4, 5, 6, 7, 8, 9, 10 or more therapeutic agents.
  • an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein and a second therapeutic agent are administered in the same dosage form. In some embodiments, an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein and a second therapeutic agent are administered in separate dosage forms.
  • an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein and a second therapeutic agent are administered concurrently (e.g., simultaneously, essentially simultaneously or within the same treatment protocol).
  • an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein and a second therapeutic agent are administered sequentially.
  • an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is administered before or after the second therapeutic agent.
  • the time period between administration of an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein and a second active agent ranges from a few minutes to several hours, depending upon the properties of each pharmaceutical agent, such as potency, solubility, bioavailability, plasma half-life and kinetic profile of the pharmaceutical agent.
  • circadian variation of the target molecule concentration determines the optimal dose interval.
  • the timing between the administration of an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein and a second active agent is about an hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about a day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about a week, about 2 weeks, about 3 weeks, about a month, or longer.
  • the co-administration of an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein results in a lower required dosage for the nHC-HA/PTX3 or rcHC-HA/PTX3 complex than the required dosage when administering an nHC-HA/PTX3 or rcHC-HA/PTX3 complex alone.
  • the co-administration of a second therapeutic agent results in a lower required dosage for the second agent than the required dosage when administering the second agent alone.
  • Combination treatment further includes periodic treatments that start and stop at various times to assist with the clinical management of the individual.
  • the combination treatment nHC-HA/PTX3 or rcHC-HA/PTX3 complex and one or more additional therapeutic agents is modified.
  • the combination treatment is modified, whereby the amount of the nHC-HA/PTX3 or rcHC-HA/PTX3 complex is increased relative to the amount of a second therapeutic agent.
  • the combination treatment is modified, whereby the amount of the nHC-HA/PTX3 or rcHC-HA/PTX3 complex is decreased relative to the amount of a second therapeutic agent.
  • the combination treatment is modified, whereby the amount of a second therapeutic agent increased relative to the amount of the nHC-HA/PTX3 or rcHC-HA/PTX3 complex.
  • the combination treatment is modified, whereby the amount of a second therapeutic agent decreased relative to the amount of the nHC-HA/PTX3 or rcHC-HA/PTX3 complex.
  • the second therapeutic agent is selected from cytotoxic agents, analgesics, anti-inflammatories, antibiotics, antimicrobial agents, anti-angiogenesis agents, chemotherapeutic agents, anti-neoplastic agents, immunotherapy, or radiation therapy.
  • the second therapeutic agent is a chemotherapeutic agent.
  • the second therapeutic agent is selected from alkylating agents, anti-metabolites, epidophyllotoxins; antineoplastic enzymes, topoisomerase inhibitors, procarbazines, mitoxantrones, platinum coordination complexes, biological response modifiers and growth inhibitors, hormonal/anti-hormonal therapeutic agents, hematopoietic growth factors, aromatase inhibitors, anti-estrogens, anti-androgens, corticosteroids, gonadorelin agonists, microtubule active agents, nitrosoureas, lipid or protein kinase targeting agents, immunomodulatory drugs (IMiDs), protein or lipid phosphatase targeting agents, anti-angiogenic agents, Akt inhibitors, IGF-I inhibitors, FGF3 modulators, mTOR inhibitors, Smac mimetics, HDAC inhibitors, agents that induce cell differentiation, bradykinin 1 receptor antagonists, angiotensin II antagonists, cyclooxygenase
  • the antimicrobial agent is an antiviral, antibacterial or antifungal agent.
  • Non-limiting exemplary antibacterial agent(s) include those classified as aminoglycosides, beta lactams, quinolones or fluoroquinolones, macrolides, sulfonamides, sulfamethaxozoles, tetracyclines, streptogramins, oxazolidinones (such as linezolid), clindamycins, lincomycins, rifamycins, glycopeptides, polymxins.
  • lipo-peptide antibiotics as well as pharmacologically acceptable sodium salts, pharmacologically acceptable calcium salts, pharmacologically acceptable potassium salts, lipid formulations, derivatives and/or analogs of the above.
  • Some exemplary classes of innate peptides or proteins are transferrins, lactoferrins, defensins, phospholipases, lysozyme, cathelicidins, serprocidins, bacteriocidal permeability increasing proteins, amphipathic alpha helical peptides, and other synthetic antimicrobial proteins.
  • the antimicrobial agent is an antiseptic agent.
  • the second therapeutic agent is selected from ARRY-797, dacarbazine (DTIC), actinomycins C 2 , C 3 , D, and F 1 , cyclophosphamide, melphalan, estramustine, maytansinol, rifamycin, streptovaricin, doxorubicin, daunorubicin, epirubicin, idarubicin, detorubicin, carminomycin, esorubicin, mitoxantrone, bleomycins A, A 2 , and B, camptothecin, Irinotecan, Topotecan, 9-aminocamptothecin, 10,11-methylenedioxycamptothecin, 9-nitrocamptothecin, bortezomib, temozolomide, TAS103, NPI0052, combretastatin, combretastatin A-2, combretastatin A-4, caliche
  • the second therapeutic agent is niacin, a fibrate, a statin, a Apo-A1 mimetic polypeptide (e.g., DF-4, Novartis), an apoA-I transcriptional up-regulator, an ACAT inhibitor, a CETP modulator, Glycoprotein (GP) IIb/IIIa receptor antagonists, P2Y12 receptor antagonists, Lp-PLA2-inhibitors, an anti-tumor necrosis factor (TNF) agent, an interleukin-1 (IL-1) receptor antagonist, an interleukin-2 (IL-2) receptor antagonist, an interleukin-6 (IL-6) receptor antagonist, an interleukin-12 (IL-12) receptor antagonist, an interleukin-17 (IL-17) receptor antagonist, an interleukin-23 (IL-23) receptor antagonist, a cytotoxic agent, an antimicrobial agent, an immunomodulatory agent, an antibiotic, a T-cell co-stimulatory blocker, a B cell depleting agent, an immunos
  • the second active agent is an anti-TGF- ⁇ antibody, an anti-TGF- ⁇ receptor blocking antibody, an anti-TNF antibody, an anti-TNF receptor blocking antibody, an anti-IL1 ⁇ antibody, an anti-IL1 ⁇ receptor blocking antibody, an anti-IL-2 antibody, an anti-IL-2 receptor blocking antibody, an anti-IL-6 antibody, an anti-IL-6 receptor blocking antibody, an anti-IL-12 antibody, an anti-IL-12 receptor blocking antibody, an anti-IL-17 antibody, anti-IL-17 receptor blocking antibody, an anti-IL-23 antibody, or an anti-IL-23 receptor blocking antibody.
  • the second active agent is alefacept, efalizumab, methotrexate, acitretin, isotretinoin, hydroxyurea, mycophenolate mofetil, sulfasalazine, 6-Thioguanine, Dovonex, Taclonex, betamethasone, tazarotene, hydroxychloroquine, sulfasalazine, etanercept, adalimumab, infliximab, abatacept, rituximab, trastuzumab, anti-CD45 monoclonal antibody AHN-12 (NCI), Iodine-131 Anti-B1 Antibody (Corixa Corp.), anti-CD66 monoclonal antibody BW 250/183 (NCI, Victoria General Hospital), anti-CD45 monoclonal antibody (NCI, Baylor College of Medicine), antibody anti-anb3 integrin (NCI), BIW-8962 (Bio
  • the second therapeutic agent is an antibiotic. In some embodiments, the second therapeutic agent is an anti-bacterial agent. In some embodiments, the second therapeutic agent is amikacin, gentamicin, kanamycin, neomycin, netilmicin, streptomycin, tobramycin, paromomycin, geldanmycin, herbimycin, loracarbef, ertapenem, doripenem, imipenem, cilastatin, meropenem, cefadroxil, cefazolin, cefalotin, cefalexin, cefaclor, cefamandole, cefoxitin, defprozil, cefuroxime, cefixime, cefdinir, cefditoren, cefoperazone, cefotaxime, cefpodoxime, ceftazidime, ceftibuten, ceftizoxime, ceftriaxone, cefepime, ceftobiprole,
  • the second therapeutic agent is a radiation therapy. In some embodiments, the second therapeutic agent is selected from x-ray therapy or proton beam therapy. In some embodiments, the radiation therapy can be external beam radiation or brachytherapy.
  • the second therapeutic agent is a targeted therapy.
  • a targeted therapy targets specific genes, proteins or tissue environment that contributes to cancer growth and survival.
  • a targeted therapy comprises one or more monoclonal antibodies.
  • a targeted therapy comprises small molecules, for example, without limitation, angiogenesis inhibitors, as described herein.
  • an targeted therapy comprises one or more monoclonal antibodies and one or more small molecules, as described herein.
  • an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is co-administered with a cell, a plurality of cells or a tissue.
  • an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is co-administered with a therapeutic cell. In some embodiments, an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is co-administered with a tissue transplant. In some embodiments, an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is co-administered with a stem cell transplant. In some embodiments, an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is co-administered with an organ transplant. In some embodiments, an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is co-administered with immune cells.
  • an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is administered concurrently (e.g., simultaneously, essentially simultaneously or within the same treatment protocol) with a tumor excision, cryoablation, or radiofrequency ablation.
  • an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is administered before or after a tumor excision, cryoablation, or radiofrequency ablation.
  • the time period between administration of an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein and the tumor excision, cryoablation, or radiofrequency ablation ranges from a few minutes to several hours, depending upon the properties of each pharmaceutical agent, such as potency, solubility, bioavailability, plasma half-life and kinetic profile of the pharmaceutical agent. In some embodiments, circadian variation of the target molecule concentration determines the optimal dose interval. In some embodiments, the timing between the administration of an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein and a second active agent is about less than an hour, less than a day, less than a week, or less than a month.
  • an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is co-administered with a tumor excision, cryoablation, or radiofrequency ablation and an immunosuppressive agent.
  • an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is co-administered with a tumor excision, cryoablation, or radiofrequency ablation and a calcineurin inhibitor (e.g., cyclosporin or tacrolimus); an mTOR inhibitor (sirolimus; everolimus); an anti-proliferative agent (azathioprine or mycophenolic acid); a corticosteroid (e.g., prednisolone or hydrocortisone); a monoclonal anti-IL-2R ⁇ receptor antibody (e.g., basiliximab or daclizumab); polyclonal anti-T-cell antibodies (e.g.,
  • a tissue is coated with an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein.
  • a plurality of stem cells are coated with an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein.
  • an organ is coated with an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein.
  • coating a tissue with an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein prevent a tissue from being acted upon by the host immune system.
  • an organ, tissue, or plurality of stem cells is contacted with an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein.
  • an organ, tissue, or plurality of stem cells is contacted with a composition comprising an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein.
  • the composition has a pH of about 7.0 to about 7.5. In some embodiments, the composition has a pH of 7.4. In some embodiments, the composition further comprises potassium, magnesium, and raffinose.
  • the composition further comprises at least one of adenosine, glutathione, allopurinol, and hydroxyethyl starch.
  • the composition is UW solution supplemented with an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein.
  • the organ, tissue, or plurality of stem cells are contacted with an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein for about 30 minutes, about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 12 hours, about 24 hours, about 36 hours, or about 48 hours.
  • the contacting occurs at a temperature that protects tissues and vascular conditioning (e.g., less than ambient temperature). In some embodiments, the contacting occurs at 4° C.
  • an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is co-administered with immune cells to a subject in need thereof.
  • the immune cells are allogenic to the subject in need thereof.
  • the immune cells are autogenic to the subject in need thereof.
  • the immune cells are genetically modified before administration to a subject in need thereof.
  • the immune cells are modified to comprise a chimeric antigen receptor (CAR T-cell therapy).
  • Example 1 Determine the Viability and Metabolic Activity of Prostate Cancer Cell-Lines after Exposure to Cryopreserved AM and UC Extracts or Purified HC-HA/PTX3 (AM)
  • Extracts or purified HC-HA/PTX3, following two or more runs of ultracentrifugation, of cryopreserved AM and UC were examined to determine if they inhibit proliferation, and reduce the overall cell metabolic activity of human prostate cancer cell-lines, i.e., PC-3 and LNCaP.
  • the WST-1 assay was used to quantify the total cell metabolic activity.
  • LNCaP tended to form aggregates ( FIG. 1 A ) while PC-3 grew as an evenly distributed single cell layer ( FIG. 1 B ).
  • Cells were harvested by 0.25% Trypsin-EDTA (cat #25200-056, Fisher Scientific) and counted by hemocytometer.
  • the total cells of LNCaP and PC-3 were 5.92 ⁇ 10 6 and 2.88 ⁇ 10 6 , respectively.
  • LNCaP 10 tubes, 5.4 ⁇ 10 5 /tube, 0.5 ml per tube ( ⁇ 1.08 ⁇ 10 6 /ml), liquid nitrogen tank: S1R4B5, and PC-3: 10 tubes, 2.6 ⁇ 10 5 /tube, 0.5 ml per tube ( ⁇ 5.2 ⁇ 10 5 /ml), liquid nitrogen tank: S1R4B5. Effects of rBTGel and HC-HA/PTX3 on Cell Metabolic Activity of LNCaP and PC-3
  • both LNCaP and PC-3 cells were treated with 0, 0.78, 1.56, 3.125, 6.25, 12.5, 25, 50, or 100 ⁇ g/ml of rBTGel (Donor #BTR161857; HA: 175 ⁇ g/ml; Protein: 165 ⁇ g/ml) or HC-HA/PTX3 (pooled fractions 3-9 of the 2nd ultracentrifugation of AM/PBS, Donor 2 for stability validation, DI: TGLP17E002) (e.g., add 11.1 ⁇ l of 10 ⁇ dose to 100 ⁇ l medium to get the exact dose).
  • cell morphology FIG. 2 A and 2 B , and FIG.
  • FIG. 3 A and 3 B was recorded by microscope images and then used for WST-1 assay (cat #10008883, Cayman Chemical Company, Ann Arbor, Mich.) for cell metabolic activity according to the manufacturer's instructions (OD450 or OD450-OD670) ( FIG. 2 C and 2 D and FIG. 3 C and 3 D ).
  • LNCaP grew as small and large cell clusters.
  • the refined BTgel at higher doses (50 and 100 ⁇ g/ml) caused most spindle-like cells to turn into round cells, but most cells were still attached and as clusters. Cell death might have also occurred based on lower cell density.
  • the similar morphological changes occurred at much lower doses of HC-HA/PTX3 (6.25 ⁇ g/ml or higher).
  • the refined BTGel was prepared in saline ( ⁇ 154 mM NaCl) whereas the HC-HA/PTX3 had been extensively dialyzed with distilled water and contained undetectable salt. Therefore, when lyophilized BTGel was added to the cell culture medium, it may have increased the salt concentration in the medium, and potentially reducing cell proliferation if it increased the salt concentration by ⁇ 30 mM or higher.
  • WST-1 assay data showed the metabolic activity of LNCaP was inhibited (p ⁇ 0.05) when the dose of the refined BTGel was at 25 ⁇ g/ml or higher ( FIG. 2 C ). Because of concern of salt effect, it is inconclusive whether the refined BTGel can inhibit the cell metabolic activity of LNCaP at these doses. In contrast, HC-HA/PTX3 at 6.25 ⁇ g/ml or higher inhibits the metabolic activity of LNCaP (by 40-85%) ( FIG. 2 C and 2 D ).
  • WST-1 assay data showed the proliferation of PC-3 was inhibited (p ⁇ 0.05) when the dose of the refined BTGel was at 6.25 ⁇ g/ml or higher ( FIG. 3 C ). Because of concern of salt effect, it is inconclusive whether the refined BTGel can inhibit the proliferation of PC-3 at these doses. In contrast, HC-HA/PTX3 at 3.125 ⁇ g/ml or higher inhibits the proliferation of PC-3 (by 8-100%) ( FIG. 3 C and 3 D ).
  • HC-HA/PTX3 did not promote PC-3 and LNCaP cancer formation, allowing for use post-prostatectomy.
  • rBTGEL was shown to have anti-cancer effects. The following example will address the effect of UC extract without potential salt effects on proliferation of prostate cancer cells.
  • BTGel at 6.25 ⁇ g/ml or higher and HC-HA/PTX3 at 3.125 ⁇ g/ml or higher inhibits the proliferation of PC-3.
  • BTGel at 25 ⁇ g/ml or higher and HC-HA/PTX3 at 6.25 ⁇ g/ml or higher inhibits the proliferation of LNCaP. Because of the concern of salt effect, it is inconclusive whether the amniotic membrane and umbilical cord (AMUC) or salt in the refined BTGel can inhibit the proliferation at these doses.
  • AMUC amniotic membrane and umbilical cord
  • a lower concentration of rBTGEL and HC-HA/PTX3 may be needed to inhibit PC-3 compared to LNCaP because PC-3 cells are known to have a quicker doubling time. Hence, the difference between the negative control and treatment groups would be greater in PC-3.
  • Example 2 Determine the Metabolic Activity of Prostate Cancer Cell-Lines after Exposure to Cryopreserved UC Extracts, HA, and Purified HC-HA/PTX3 (AM)
  • Example 1 show HC-HA/PTX3 reduces the metabolic activity of both prostate cell lines at concentrations as low as 6.25 ⁇ g/ml in both cell types.
  • rBTGEL was shown to inhibit the activity at 25 ⁇ g/ml and above. The following study was performed to rule out the possibility that salt concentration in the higher doses of rBTGel was confounding the data interpretation.
  • UC extract in water was tested using the same WST-1 assay.
  • HC-HA/PTX3 was tested to compare to UC results.
  • HA was used as a control group.
  • a series of doses were tested based on the HA ⁇ g/ml.
  • purified HC-HA/PTX3 pooled fractions 3-9 of the 2nd ultracentrifugation of AM/PB S, Donor 2 for stability validation, DI: TGLP17E002, prepared on Sep. 19, 2017
  • WST-1 assay data showed the metabolic activity of LNCaP cells was significantly inhibited (p ⁇ 0.05) when treated with UC extract ( ⁇ 100 ⁇ g/ml) and HC-HA/PTX3 ( ⁇ 6.25 ⁇ g/ml) but not HA (See FIG. 4 A ).
  • the metabolic activity in PC-3 cells was significantly inhibited by UC extract ( ⁇ 200 ⁇ g/ml) and HC-HA/PTX3 ( ⁇ 1.56 ⁇ g/ml) but not HA (See FIG. 4 B ).
  • Cell death was markedly noted with microscopy in both LNCaP ( FIGS. 5 A- 5 C ) and PC3 ( FIGS. 6 A- 6 C ) cells with HC-HA/PTX3 at concentrations ⁇ 25 ⁇ g/ml.
  • WST-1 assay data showed the metabolic activity of LNCaP cells was significantly inhibited (p ⁇ 0.05) when treated with UC extract ( ⁇ 100 ⁇ g/ml) and HC-HA/PTX3 ( ⁇ 6.25 ⁇ g/ml) but not HA.
  • the metabolic activity in PC-3 cells was significantly inhibited by UC extract ( ⁇ 200 ⁇ g/ml) and HC-HA/PTX3 ( ⁇ 1.56 ⁇ g/ml) but not HA.
  • Cell death was markedly noted with microscopy in both LNCaP and PC3 cells with HC-HA/PTX3 at concentrations ⁇ 25 ⁇ g/ml.
  • LNCaP attachment to a surface is needed for viability and proliferation, and detachment induces cell death through the process of anoikis.
  • the literature has compared the effect of LNCaP grown on different coating reagents (poly-1-lysine, poly-1-ornithine, collagen from human placenta type IV, fibronectin, and laminin) and showed laminin and collagen type IV promoted cell aggregation ( FIG. 7 , taken at 24 hours). This aggregation is similar to the morphology seen in the experiments provided herein and may suggest HC-HA/PTX3 reduces the LNCaP cell-surface attachment.
  • liquid overlay technique is commonly used in this field to induce aggregation/spheroids by culturing cells on surfaces with non-adherent properties and thus cell-cell interactions are more prominent than those established between the cells and the surface. Consequently, cells aggregate leading to the formation of spheroids in 1-3 days, for the majority of the cell lines.
  • WST-1 assay data showed the metabolic activity of LNCaP cells was significantly inhibited (p ⁇ 0.05) when treated with UC extract ( ⁇ 100 ⁇ g/ml) and HC-HA/PTX3 ( ⁇ 6.25 ⁇ g/ml) but not HA.
  • the metabolic activity in PC-3 cells was significantly inhibited by UC extract ( ⁇ 200 ⁇ g/ml) and HC-HA/PTX3 ( ⁇ 1.56 ⁇ g/ml) but not HA.
  • Cell death was markedly noted with microscopy in both LNCaP and PC3 cells with HC-HA/PTX3 at concentrations ⁇ 25 ⁇ g/ml.
  • the prostate epithelium is composed of two histologically distinct layers: the secretory luminal layer and basal cell layer.
  • Human normal prostate epithelial basal and luminal cells were used.
  • Human normal prostate epithelial basal cells (PrEC) were obtained from Clonetics-BioWhittaker, Inc. (Walkersville, Md., USA), and were cultured in prostate epithelial basal medium with PrEgM BulletKit (both from Clonetics) containing supplements and growth factors (BPE, hydrocortisone, hEGF, epinephrine, insulin, triiodothyronine, transferrin, gentamicin/amphotericin B, and retinoic acid).
  • BPE growth factors
  • Human normal prostate luminal PNT2 Cell Line was purchased from Sigma (cat 95012613). The cell line was established by immortalization of normal adult prostatic epithelial cells by transfection with a plasmid containing SV40 genome with a defective replication origin.
  • the PNT2 cells were cultured in in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS), L-glutamine (2 mM), penicillin (100 U/ml), and streptomycin (100 ⁇ g/ml) (RPMI 1640 complete medium) at 37° C. in a humidified incubator in an atmosphere of 5% CO2 in air. Upon 70-80% confluency, cells were harvested by 0.25% (w/v) Trypsin-0.53 mM EDTA solution and were stored in 95% complete medium and 5% DMSO at liquid nitrogen vapor phase in aliquots.
  • FBS fetal bovine serum
  • L-glutamine 2 mM
  • penicillin 100 U/ml
  • Cells were treated with a series of doses of purified HC-HA/PTX3 or HA and were kept at 37° C. for 48 h.
  • the cell morphology was recorded under microscope (bright-field in 10 ⁇ and 20 ⁇ magnifications) before treatment and after 24 h and 48 h of treatment.
  • the metabolic activity of each cell-line was measured by WST-1 (cat #10008883, Cayman Chemical Company, Ann Arbor, Mich.) following the manufacturer's instructions (OD450 or OD450-OD670) at 48 h.
  • PrEC Human normal prostate epithelial basal cells grow faster than human normal prostate luminal cells (PNT2). For PrEC doubling time is 18-24 h, while for PNT2, it is longer about 36-48 h.
  • PrEC cells typically proliferated fast and were well attached on the surface. Usually cells were well adhered to each other.
  • Metabolic activity (%) was evaluated in the normal human primary prostate epithelial cells PrEC and normal human prostate cell line PNT2 by WST-1 assay after 48 hr. incubation with different concentrations (0.78, 3.125, 6.25, 12.5, 25, 50, 100 ⁇ g/ml) of HC-HA/PTX3 or HA. P-value calculated by 2-tailed t-test with respect to the untreated samples.
  • FIGS. 11 A- 11 B and FIGS. 12 A- 12 B show comparative analysis of the metabolic activity (%) evaluated in the normal primary prostate epithelial cells (PrEC) and cell lines (PNT2) and prostate cancer cell lines: PC3 and LNCaP by WST-1 assay after 48 hr incubation with different concentrations (0.78, 3.125, 6.25, 12.5, 25, 50, 100 ⁇ g/ml) of HC-HA/PTX3 and HA.
  • Semi-logarithmic regression analysis demonstrated, HC-HA/PTX3 inhibited metabolic activity of all types of prostate cells linearly in a dose-dependent manner while HA did not have any significant effect * (p ⁇ 0.05); ** (p ⁇ 0.01); *** (p ⁇ 0.001).
  • HC-HA/PTX3 inhibited cell metabolic activity of all types of human prostate cells/cell lines, including normal and cancer in dose dependent manner while HMW-HA had no significant effect.
  • concentrations up to 25 ⁇ g/ml PNT2 cell line showed less sensitivity towards HC-HA/PTX3 (cell metabolic activity: 60%; p ⁇ 0.001) while the metabolic activity of the cancer cell lines (both PC3 and LNCaP) were reduced below 25% (p ⁇ 0.001).
  • HC-HA/PTX3 regulated metabolic activity of two prostate cancer cell-lines (PC-3 and LNCaP).
  • PC-3 and LNCaP prostate cancer cell-lines
  • HC-HA/PTX3 and HMW-HA as control
  • A-375 melanoma
  • A549 lung cancer
  • MCF-7 breast cancer
  • HT-29 colon adenocarcinoma
  • Example 1 It was noted in Example 1, that the HC-HA/PTX3-response curve plateaus at concentrations of 50 ⁇ g/ml and above. There was no significant variation in metabolic activity between 50 ⁇ g/ml and 100 ⁇ g/ml concentration of HC-HA/PTX3. As such, the maximum concentration of HC-HA/PTX3 was maintained at 100 ⁇ g/ml.
  • each well of a 96-well plate was seeded with 3200 cells in 100 ⁇ l culture medium.
  • Cells were treated with the following concentrations of HC-HA/PTX3 and HA (as control): 0.78, 1.56, 3.125, 6.25, 12.5, 25, 50, 100 ( ⁇ g/ml) in triplicate.
  • Two untreated samples were used: untreated without WST-1 reagent and untreated with WST-1 reagent (as control for WST-1 assay). To avoid pipetting error, cells were diluted to a final volume of 8 ml in culture medium as shown in Table 2.
  • A-375 (melanoma): Normally, cells proliferate fast and are well attached on the surface.
  • HC-HA/PTX3 treatment After 24 h: at 25 ( ⁇ g/ml): cells tended to be spindle shaped. At 50 ( ⁇ g/ml): cells turned spindle shaped. At 100 ( ⁇ g/ml): cells were round and dead. After 48 h: at 6.25 ( ⁇ g/ml): cells showed slightly lose intercellular adherence. At 12.5 ( ⁇ g/ml): Cells lost more intercellular adherence. Cells tended to grow individually rather than in clump. At 25 ( ⁇ g/ml): >80% cells were shrinking, spindle shaped, remaining cells were round. At 50 ( ⁇ g/ml): >60% cells were dead and appeared round, remaining cells were spindle-shaped. At 100 ( ⁇ g/ml): all cells appeared as round and were dead. See, FIG. 13 .
  • HA treatment No significant effect of HA observed.
  • HT-29 colon cancer: cells are normally round and grow in aggregate. Each aggregate appears like a ball.
  • HC-HA/PTX3 treatment After 24 h: at 50 ( ⁇ g/ml): cells lost adhesion and were isolated. At 100 ( ⁇ g/ml): cells appeared as round bead-like shape and survived less. After 48 h: at 1.56 ( ⁇ g/ml): intercellular adherence was gradually lost. At 6.25 ( ⁇ g/ml): ⁇ 10 cells adhered to each other in each clump. Cells lost intercellular adherence more. At 25 ( ⁇ g/ml): cells tended to grow in single rather than in aggregate. At 50 ( ⁇ g/ml): cells grew in single. At 100 ( ⁇ g/ml): single cells round shaped and survived less. See, FIG. 14 .
  • HA treatment No significant effect of HA observed.
  • A549 lung cancer: Normally cells are epithelial and proliferate quickly.
  • HC-HA/PTX3 treatment After 24 h: at 25 ( ⁇ g/ml): cells morphology slightly changed to spindle shaped. At 50 ( ⁇ g/ml) cells tend to be more spindle shape. At 100 ( ⁇ g/ml): All cells are not dead. Dead cells are round. After 48 h: at 12.5 ( ⁇ g/ml): cell morphology slightly changed to spindle shape. At 50 ( ⁇ g/ml): cells tend to be more spindle shape. At 100 ( ⁇ g/ml): All cells are not dead. Dead cells are round. See, FIG. 15 .
  • HA treatment No significant effect of HA observed.
  • MCF-7 breast cancer: Normally cells are epithelial. Slow growing. Adhered to each other. Grow in aggregate.
  • HC-HA/PTX3 treatment After 24 h: No effect until 50 ⁇ g/ml. At 100 ( ⁇ g/ml): Round bead like cells dead. After 48 h, the effect was same. See, FIG. 16 .
  • HA treatment No significant effect of HA observed.
  • HT-29 HT-29 cell metabolism decreased significantly in a dose-dependent manner. After 48 h treatment with HC-HA/PTX3, cell metabolic activity changed significantly from 6.25 ⁇ g/ml onwards. No significant effect of HA was observed. ( FIG. 17 B )
  • MCF-7 showed the least sensitivity to HC-HA/PTX3. Significant effect of HC-HA/PTX3 could be observed at a high concentration (50 ⁇ g/ml) but the effect was not as strong as observed for other cell lines like A375 and HT-29. ( FIG. 17 C )
  • HC-HA/PTX3 inhibited cellular aggregation, intercellular junction, cell shape and cell adhesion, thus cell metabolic activity of all the 4 types of cancer cells tested in a dose-dependent manner.
  • Hyaluronan had no significant effect on the cellular morphology and metabolic activity of all four cell types.
  • HC-HA/PTX3 affects the morphology and metabolic activity of normal prostate epithelial cells similar to those of tumor cells. To address if such an effect also applies to normal mesenchymal cells, it was examined whether HC-HA/PTX3 also exerts similar effects on the morphology and the metabolic activity of a series of mesenchymal cells.
  • LNC limbal niche cells
  • HTM human trabecular meshwork
  • HCF human corneal fibroblast
  • cells were seeded in each well in 1000 ⁇ l culture medium. After cells achieved sufficient confluency, they were trypsinized followed by brief centrifugation at 200 g for 5 mins. Medium was removed and the cell pellet was supplemented with fresh culture medium. Selected wells of a 96-well plate were coated with 50 ⁇ l 5% matrigel and incubated at 37° C. for 1 hr. Afterwards, LNC and HTM cells were seeded in matrigel-coated 96-well plates and kept at 37° C. for overnight incubation. In each well, 3200 cells were seeded in 100 ⁇ l culture medium.
  • HC-HA/PTX3 purified HC-HA/PTX3 and HA.
  • concentration of HC-HA/PTX3 and HA was calculated by serial dilution as done in Example 3.
  • the cell morphology was recorded after 15-30 mins, 1 h, 5 h, 24 h and 48 h of treatments respectively. After 48 h incubation, the cell metabolic activity was measured by WST-1 (cat #10008883, Cayman Chemical Company, Ann Arbor, Mich.) according to the manufacturer's instructions (OD450 or OD450-OD670).
  • FIG. 18 B shows representative bright-field microscopic image (scale bar 50 ⁇ m) of LNC (limbal niche cells) after 48 h incubation with 100 ⁇ g/ml of HC-HA/PTX3 or HMW-HA.
  • FIG. 19 shows metabolic activity (%) evaluated in limbal niche cells by WST-1 assay after 48 h incubation with different concentrations (1.56, 3.125, 6.25, 12.5, 25, 50, 100 ⁇ g/ml) of HC-HA/PTX3 and HA. p-value calculated by 2-tailed Student's t-test with respect to the untreated samples.
  • HTM Human trabecular meshwork
  • FIG. 20 A and 20 B show representative bright-field microscopic images (scale bar 50 ⁇ m) of HTM (human trabecular meshwork) cells after treatment with different concentrations of HC-HA/PTX3 ( FIG. 20 A ) and HMW-HA ( FIG. 20 B ) for different time points: 15-30 mins, 1 hr, 5 hr, 24 hr and 48 hr. respectively.
  • FIG. 21 shows metabolic activity (%) evaluated in human trabecular meshwork cells by WST-1 assay after 48 hr. incubation with different concentrations (1.56, 3.125, 6.25, 12.5, 25, 50, 100 ⁇ g/ml) of HC-HA/PTX3 and HA. p-value calculated by 2-tailed Student's t-test with respect to the untreated samples. As shown, HC-HA/PTX3 and HMW-HA had little effect on HTM.
  • FIG. 22 A and 22 B shows representative brightfield microscopic images (scale bar 50 ⁇ m) of human corneal fibroblast (HCF) cells after treated with different concentrations of HC-HA/PTX3 ( FIG. 22 A ) and HMW-HA ( FIG. 22 B ) for different time points: 15-30 mins, 1 hr, 5 hr, 24 hr and 48 hr. respectively.
  • FIG. 23 shows metabolic activity (%) evaluated in human corneal fibroblast cells by WST-1 assay after 48 hr. incubation with different concentrations (1.56, 3.125, 6.25, 12.5, 25, 50, 100 ⁇ g/ml) of HC-HA/PTX3 and HA. p-value calculated by 2-tailed Student's t-test with respect to the untreated samples.
  • FIGS. 24 A and 24 B show comparative analysis of the metabolic activity (%) evaluated in three types of human normal primary mesenchymal cells: HCF, HTM & LNC as evaluated by WST-1 assay after 48 hr. incubation with different concentrations (1.56, 3.125, 6.25, 12.5, 25, 50, 100 ⁇ g/ml) of HC-HA/PTX3 ( FIG. 24 A ) and HA ( FIG. 24 B ).
  • p-value calculated by 2-tailed Student's t-test with respect to the untreated samples. *denotes p ⁇ 0.05.
  • Example 6 Determine the Role of HC-HA/PTX3 in Suppressing Cell Proliferation in Human Normal and Cancer Cells
  • Example 4 the metabolic activity (measured by WST-1) and morphology of A375 (melanoma) cells are sensitive to HC-HA/PTX3. As the concentration of HC-HA/PTX3 increased, metabolic activity was inhibited, and the cell morphology was changed from epithelial to spindle shape and then became round losing intercellular and cell-matrix adhesion. Since cell metabolic activity is directly proportional to the cell proliferation rate, the role of HC-HA/PTX3 in suppressing cell proliferation by quantifying DNA content of the proliferating cells will be determined. 5-bromo-2′-deoxyuridine (BrdU) incorporated into cellular DNA during cell proliferation will be detected using an anti-BrdU antibody by the BrdU Cell Proliferation Assay Kit (Cat #6813; Cell Signaling Technology, USA).
  • RhdU 5-bromo-2′-deoxyuridine
  • Example 4 wherein A375 cells showed significant morphological change starting from 25 ⁇ g/ml onwards of HC-HA/PTX3 after 24 hr treatment, cells were treated with HC-HA/PTX3 at following concentrations: 0, 25, 50 and 100 ⁇ g/ml for 24 hrs. 100 ⁇ g/ml of HA was used as control due to the lack of significant effect on cell morphology and metabolic activity as shown in Example 5. Based on this pilot study, the proliferation assay protocol was optimized and applied to assess the effect of HC-HA/PTX3 on human prostate cells (normal & cancer). Test groups are shown in Table 4.
  • A375 cells were seeded in 96-well plate (3200 cells in 100 ⁇ l culture medium/well) and were incubated for overnight. Cells were treated with HC-HA/PTX3 and HA at aforementioned concentrations for 48 hrs. 10 ⁇ l of 10 ⁇ BrdU solution in each well and the cells were placed in incubator for 4 h. Removing medium, fixing/denaturing solution was added 100 ⁇ l/well for 30 min. Removing the solution, 1 ⁇ detection antibody solution was added 100 ⁇ l/well for 1 h. The solution was removed and washed properly with wash buffer for three times and 1 ⁇ HRP-conjugated secondary antibody solution was added 100 ⁇ l/well for 30 min at RT. Solution was removed and washed properly with wash buffer for three times and TMB substrate was added 100 ⁇ l/well for 30 min at RT. STOP solution was added 100 ⁇ L/well and absorbance was read at 450 nm.
  • PrEC, PNT2, PC-3 and LNCaP cells were seeded at 3.2 ⁇ 10 3 cells/well in a 96-well plate and incubated overnight. Cells were then treated with five concentrations of HC-HA/PTX3 (1.56, 3.13, 6.25, 12.5 & 25 ⁇ g/ml) and 100 ⁇ g/ml HMW-HA for 48 hrs in triplicate, as shown in Table 5. Finally, 10 ⁇ M BrdU was added to the well and cells were incubated for 4 hr. Medium was removed and fixing/ denaturing solution added 100 ⁇ l/well for 30 min. Solution was removed and 1 ⁇ detection antibody solution was added and 1 ⁇ HRP-conjugated secondary antibody solution was added 100 ⁇ l/well for 30 min at RT. Solution was removed and washed properly with wash buffer three times and TMB substrate was added 100 ⁇ l/well for 30 min at RT. STOP solution was added 100 ⁇ L/well and absorbance read at 450 nm.
  • FIG. 27 A and 27 B show HC-HA/PTX3 inhibited PrEC cell proliferation in a dose-dependent manner while HMW-HA ( FIG. 27 C ) had no significant effect on cell proliferation as detected by BrdU Cell Proliferation Assay Kit #6813 (Cell Signaling, USA).
  • FIG. 27 A shows bright-field images of PrEC cell morphology in two magnifications (10 ⁇ & 20 ⁇ );
  • FIGS. 27 B and 27 C show BrdU cell proliferation assay curve. Statistical significance (p value) calculated from Student's t-test.
  • HC-HA/PTX3 inhibited PNT2 cell proliferation in a dose-dependent manner while HMW-HA had no significant effect on cell proliferation as detected by BrdU Cell Proliferation Assay Kit #6813 (Cell Signaling, USA).
  • PNT2 cells were seeded at 3.2 ⁇ 10 3 cells/well in a 96-well plate and incubated overnight. Cells were then treated with five concentrations of HC-HA/PTX3 (1.56, 3.13, 6.25, 12.5 & 25 ⁇ g/ml) and two concentrations of HMW-HA (25 & 100 ⁇ g/ml) for 48 hrs in triplicate. Finally, 10 ⁇ M BrdU was added to the well and cells were incubated for 4 hr.
  • HC-HA/PTX3 but not HA had anti-proliferative effect on PC-3 prostate cancer cell line as observed in Example 7.
  • HC-HA/PTX3 inhibited PC3 cell proliferation in dose-dependent manner while HMW-HA had no significant effect on cell proliferation as detected by BrdU Cell Proliferation Assay Kit #6813 (Cell Signaling, USA).
  • PC3 cells were seeded at 3.2 ⁇ 10 3 cells/well in a 96-well plate and incubated overnight. Cells were then treated with five concentrations of HC-HA/PTX3 (1.56, 3.13, 6.25, 12.5 & 25 ⁇ g/ml) and two concentrations of HMW-HA (25 & 100 ⁇ g/ml) for 48 hrs in triplicate. Finally, 10 ⁇ M BrdU was added to the well and cells were incubated for 4 hr.
  • FIG. 29 A shows bright-field images of PC3 cell morphology.
  • FIG. 29 B shows BrdU cell proliferation assay curve.
  • LNCaP Under treatment with HC-HA/PTX3, the cell morphology of LNCaP was changed in a dose-dependent manner. Surprisingly, despite being a cancer cell, LNCaP did not proliferate as fast as PC-3 or PNT2 or PrEC cells. This observation was well corroborated in the BrdU data of all the cell types. Under untreated condition, O.D. values of LNCaP cells came 0.5 while for rest other cells it was >0.75. Interestingly, for LNCaP, cells kept on growing in aggregate even at high concentration of HC-HA/PTX3, but their morphology changed, thus implicating, the effect of HC-HA/PTX3 more on cell-matrix attachment rather than cell-cell adhesion.
  • HC-HA/PTX3 inhibited LNCaP cell proliferation in dose-dependent manner while HMW-HA had no significant effect on cell proliferation as detected by BrdU Cell Proliferation Assay Kit #6813 (Cell Signaling, USA).
  • LNCaP cells were seeded at 3.2 ⁇ 10 3 cells/well in a 96-well plate and incubated overnight. Cells were then treated with five concentrations of HC-HA/PTX3 (1.56, 3.13, 6.25, 12.5 & 25 ⁇ g/ml) and two concentrations of HMW-HA (25 & 100 ⁇ g/ml) for 48 hrs in triplicate. Finally, 10 ⁇ M BrdU was added to the well and cells were incubated for 4 hr.
  • FIG. 30 A shows bright-field images of LNCaP cell morphology.
  • FIG. 30 B shows BrdU cell proliferation assay curve.

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CN115397841A (zh) 2022-11-25
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AU2021219772A1 (en) 2022-09-01
KR20220143689A (ko) 2022-10-25
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